Name: endless worms most beautiful: current methods for using nematodes to study evolutionary developmental biology
Uploaded: 2022-08-10T14:00:00
Description: Read this Scientific Article Protocol about Endless Worms Most Beautiful: Current Methods For Using Nematodes To Study Evolutionary Developmental Biology at JoVE.com

The authors acknowledge the funding from the National Institutes of Health (# R01GM143771 and # R01HD081266).

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+powerful+new+quantitative+genetics+platform,+combining+Caenorhabditis+elegans+high-throughput+fitness+assays+with+a+large+collection+of+recombinant+strains.">A powerful new quantitative genetics platform, combining Caenorhabditis elegans high-throughput fitness assays with a large collection of recombinant strains.</a> G3: Genes|Genomes|Genetics. 5 (5), 911 (2015).</li><li>McGrath, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mapping+of+a+digenic+behavioral+trait+implicates+globin+variation+in+C.+elegans+sensory+behaviors.">Quantitative mapping of a digenic behavioral trait implicates globin variation in C. elegans sensory behaviors.</a> Neuron. 61 (5), 692-699 (2009).</li><li>Zhao, Z., Boyle, T. J., Bao, Z., Murray, J. I., Mericle, B., Waterston, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+embryonic+cell+lineage+between+Caenorhabditis+briggsae+and+Caenorhabditis+elegans.">Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans.</a> Developmental Biology. 314 (1), 93-99 (2008).</li><li>Levin, M., Hashimshony, T., Wagner, F., Yanai, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+milestones+punctuate+gene+expression+in+the+Caenorhabditis+embryo.">Developmental milestones punctuate gene expression in the Caenorhabditis embryo.</a> Developmental Cell. 22 (5), 1101-1108 (2012).</li><li>Ewe, C. K., Torres Cleuren, Y. N., Rothman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+developmental+system+drift+in+the+endoderm+gene+regulatory+network+of+Caenorhabditis+and+other+nematodes.">Evolution and developmental system drift in the endoderm gene regulatory network of Caenorhabditis and other nematodes.</a> Frontiers in Cell and Developmental Biology. 8, 170 (2020).</li><li>True, J. R., Haag, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+system+drift+and+flexibility+in+evolutionary+trajectories.">Developmental system drift and flexibility in evolutionary trajectories.</a> Evolution and Development. 3 (2), 109-119 (2001).</li><li>Kanzaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+genome+of+a+newly+discovered+sibling+species+of+Caenorhabditis+elegans.">Biology and genome of a newly discovered sibling species of Caenorhabditis elegans.</a> Nature Communications. 9 (1), 1-12 (2018).</li><li>Tintori, S. C., Sloat, S. A., Rockman, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+wild+nematodes+by+Baermann+funnel.">Rapid isolation of wild nematodes by Baermann funnel.</a> Journal of Visualized Experiments. (179), e63287 (2022).</li><li>Crombie, T. A., Tanny, R. E., Buchanan, C. M., Roberto, N. M., Andersen, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+scalable+approach+to+perform+ecological+surveys+of+selfing+Caenorhabditis+nematodes.">A highly scalable approach to perform ecological surveys of selfing Caenorhabditis nematodes.</a> Journal of Visualized Experiments. (181), e63486 (2022).</li><li>Andersen, E. C., Rockman, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+genetic+variation+as+a+tool+for+discovery+in+Caenorhabditis+nematodes.">Natural genetic variation as a tool for discovery in Caenorhabditis nematodes.</a> Genetics. 220 (1), 156 (2022).</li><li>Lee, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+basis+of+natural+variation+in+a+phoretic+behavior.">The genetic basis of natural variation in a phoretic behavior.</a> Nature Communications. 8 (1), 1-7 (2017).</li><li>Jovelin, R., Ajie, B. C., Phillips, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+and+quantitative+variation+for+chemosensory+behaviour+in+the+nematode+genus+Caenorhabditis.">Molecular evolution and quantitative variation for chemosensory behaviour in the nematode genus Caenorhabditis.</a> Molecular Ecology. 12 (5), 1325-1337 (2003).</li><li>Ackley, C., Washiashi, L., Krishnamurthy, R., Rothman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+Gravitaxis+assay+of+Caenorhabditis+Dauer+larvae.">Large-scale Gravitaxis assay of Caenorhabditis Dauer larvae.</a> Journal of Visualized Experiments. (183), e64062 (2022).</li><li>Ackley, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+mechanosensory+systems+are+required+for+negative+gravitaxis+in+C.+elegans.">Parallel mechanosensory systems are required for negative gravitaxis in C. elegans.</a> bioRxiv. , (2022).</li><li>Kaletsky, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+adult+Caenorhabditis+elegans+cells+reveals+tissue-specific+gene+and+isoform+expression.">Transcriptome analysis of adult Caenorhabditis elegans cells reveals tissue-specific gene and isoform expression.</a> PLOS Genetics. 14 (8), 1007559 (2018).</li><li>Woronik, A., Kiontke, K., Jallad, R. S., Herrera, R. A., Fitch, D. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+for+species-agnostic+single-tissue+applications.">Laser microdissection for species-agnostic single-tissue applications.</a> Journal of Visualized Experiments. (181), e63666 (2022).</li><li>Rosso, M. N., Jones, J. T., Abad, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+and+functional+genomics+in+plant+parasitic+nematodes.">RNAi and functional genomics in plant parasitic nematodes.</a> Annual Review of Phytopathology. 47, 207-232 (2009).</li><li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ditylenchus+destructor+genome+provides+new+insights+into+the+evolution+of+plant+parasitic+nematodes.">The Ditylenchus destructor genome provides new insights into the evolution of plant parasitic nematodes.</a> Proceedings. Biological Sciences. 283 (1835), 20160942 (2016).</li><li>Cammalleri, S. R., Knox, J., Roy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+screening+the+plant+parasitic+nematode+Ditylenchus+dipsaci.">Culturing and screening the plant parasitic nematode Ditylenchus dipsaci.</a> Journal of Visualized Experiments. (179), e63438 (2022).</li><li>Mimee, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+draft+genome+of+Ditylenchus+dipsaci.">The draft genome of Ditylenchus dipsaci.</a> Journal of Nematology. 51 (1), 1-3 (2019).</li><li>Heryanto, C., Ratnappan, R., O&#8217;halloran, D. M., Hawdon, J. M., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+genetically+manipulating+entomopathogenic+nematodes.">Culturing and genetically manipulating entomopathogenic nematodes.</a> Journal of Visualized Experiments. (181), e63885 (2022).</li><li>Macchietto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptomics+of+Steinernema+and+Caenorhabditis+single+embryos+reveals+orthologous+gene+expression+convergence+during+late+embryogenesis.">Comparative transcriptomics of Steinernema and Caenorhabditis single embryos reveals orthologous gene expression convergence during late embryogenesis.</a> Genome Biology and Evolution. 9 (10), 2681 (2017).</li></ol>

The authors have nothing to disclose.

In the 1960s, Sydney Brenner pioneered the use of Caenorhabditis elegans, specifically the N2 strain isolated from Bristol, UK, to study animal neurobiology and development1. Since then, seminal discoveries with this laboratory reference strain have been instrumental for unraveling the&#160;regulation of fundamental processes such as programmed cell death2, organogenesis3,4,5, aging6,7, gene regulation by noncoding RNAs8,9,10,11, the description of complete sexually dimorphic neural connectomes12, and many other phenomena in molecular, cell, and developmental biology. Complemented by several &#8220;omic&#8221; studies at single-cell resolution13,14, N2 is arguably the most completely described metazoan. The wealth of knowledge gained from studying N2 over the past six decades has provided a springboard for exploring the evolutionary diversification of the machinery that modulates development in genetically distinct C. elegans wild isotypes, as well as in other nematode species. To date, more than 500 C. elegans isotypes, each with a unique haplotype, have been isolated from all around the world15. This advance has greatly facilitated the identification of the genetic factors responsible for variation in quantitative traits16, including laboratory-derived alleles in many genes that modify an array of behavioral and developmental traits in N2 (e.g., references17,18,19,20). Hence, studying wild worms can provide important insights into how animals react to their environment in ways that influence development, physiology, and behaviors and also illuminate the evolutionary trajectories of the animals. Many species in the Caenorhabditis genus appear virtually indistinguishable morphologically from C. elegans21,22, providing a strong platform to study the molecular mechanism of developmental system drift23,24; however, it is interesting to note that the morphology of C. elegans, which inhabits rotting plant material, differs substantially from its closest known sibling, C. inopinata, found only on figs, likely as the result of niche adaption25. Intra- and interspecies comparative studies will deepen our understanding of the evolutionary history of these animals. With the goal of promoting the use of the rich natural resource that nematodes offer to probe important questions in evolutionary developmental and behavioral biology, this collection of articles provides useful techniques for isolating, culturing, and studying wild worms and nematode species outside of the C. elegans species.

In this Methods Collection, Tintori et al.26 and Crombie et al.27 provide complementary approaches to isolating wild nematodes. Tintori et al. describe&#160;a simple, cost-effective method for isolating nematodes in the field using the Baermann funnel. This approach allows researchers, in an unbiased way, to collect nearly the entire nematode population from the substrates (e.g., rotting fruit, flowers, and stems). In contrast, Crombie et al. present a method that enriches self-fertilizing Caenorhabditis species such as C. elegans, C. briggsae, and C. tropicalis. Additionally, they provide detailed instructions on the use of the Fulcrum app and R software to effectively track and organize the nematode samples, as well as the corresponding ecological data. Studying nematodes in their natural context will provide important insights into their ecology, which shapes the complex traits in these species.

Nematodes exhibit intra- and interspecies variation in behaviors that facilitate&#160;niche adaptation, providing an excellent paradigm to study the genetic basis of behavioral plasticity and neurodivergence28,29,30. Ackley et al.31 describe a robust assay to study gravitaxis, which may serve as a dispersal mechanism, in dauer larvae of different Caenorhabditis species. As gravitactic behavior may be influenced by other sensory inputs32, Ackley et al.31 provide a simple and cost-effective way to shield the animals from light and electromagnetic fields, thereby preventing the known interference of these environmental influences on gravitactic behavior. This assay will be useful for studying the molecular mechanism of sensory integration and the evolution of dispersal strategies in nematodes.

The development of single-cell RNA sequencing techniques in the past decade has revolutionized transcriptomic analyses, enabling researchers to study cellular heterogeneity in multicellular organisms at high resolution. In C. elegans, the isolation of different cell types relies on the expression of tissue-specific fluorescent markers and FACS33, which limit its application to other nematode species for which transgenic tools are not available. Woronik et al.34 successfully deploy&#160;laser microdissection to isolate tissues from worms in the absence of cellular markers. They isolate&#160;tail tips from C. elegans males and hermaphrodites and perform&#160;RNA-seq using CEL-Seq2 to investigate the mechanism of tail morphogenesis; however, it is worth noting that the method described can be readily adopted for other tissue types in different nematode species, allowing comparative transcriptome analyses in a tissue-specific context.

C. elegans and many parasitic nematodes contain similar structures. In addition, many major signaling systems and developmental pathways are conserved between species35,36. The stem and bulb nematode, Ditylenchus dipsaci, is a plant parasitic nematode affecting over 500 plant species worldwide that causes devastating loss of food crops. Cammalleri et al.37 describe an effective method to propagate a large population of D. dipsaci in pea plants. The authors also provide a detailed protocol for identifying small-molecule candidates that may serve as nematocides by impairing the worms&#8217; mobility. The ease of laboratory cultivation coupled with the recently available genome sequence38 make&#160;D. dipsaci a useful model for comparative studies with C. elegans, making it possible to uncover novel aspects of its biology and to develop effective strategies for biocontrol.

Lastly, Heryanto et al.39 provide a method to propagate Heterorhabditis bacteriophora and Steinernema carpocapsae, which are entomophathogenic worms that infect insects and exhibit mutualistic association with the bacteria in their guts. Heryanto et al. also demonstrate methods for RNAi in H. bacteriophora by microinjecting dsRNA into the gonad. A previous study showed that Steinernema embryos are slow-developing&#160;and that maternal-to-zygotic transition occurs earlier during embryogenesis in Steinernema than in Caenorhabditis species40. Further comparative studies using the genetic tools described by Heryanto et al. 39 promise to reveal the basis of the evolutionary diversification of developmental programs in these distantly related species, as well as novel insights into the evolution of parasitism.

The articles in this Methods Collection provide samplers of the powerful system nematodes offer for future comparative and mechanistic studies. It is of great interest to understand how diverse biological processes are tuned during evolution to manifest Charles Darwin&#8217;s &#8220;endless forms most beautiful and most wonderful.&#8221;

endless worms most beautiful: current methods for using nematodes to study evolutionary developmental biology

ARTICLES DISCUSSED:
Tintori, S. C., Sloat, S. A., Rockman, M. V. Rapid isolation of wild nematodes by baermann funnel. Journal of Visualized Experiments. (179) (2022).
Crombie, T. A., Tanny, R. E., Buchanan, C. M., Roberto, N. M., Andersen, E. C. A highly scalable approach to perform ecological surveys of selfing Caenorhabditis nematodes. Journal of Visualized Experiments. (181) (2022).
Ackley, C., Washiashi, L., Krishnamurthy, R., Rothman, J. H. Large-scale gravitaxis assay of Caenorhabditis Dauer larvae. Journal of Visualized Experiments. (183) (2022).
Woronik, A., Kiontke, K., Jallad, R. S., Herrera, R. A., Fitch, D. H. A. Laser microdissection for species-agnostic single-tissue applications. Journal of Visualized Experiments. (181) (2022).
Cammalleri, S. R., Knox, J., Roy, P. J. Culturing and screening the plant parasitic nematode Ditylenchus dipsaci. Journal of Visualized Experiments. (179) (2022).
Heryanto, C., Ratnappan, R., O&#8217;halloran, D. M., Hawdon, J. M., Eleftherianos, I. Culturing and genetically manipulating entomopathogenic nematodes. Journal of Visualized Experiments. (181) (2022).

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Endless Worms Most Beautiful: Current Methods For Using Nematodes To Study Evolutionary Developmental Biology

ARTICLES DISCUSSED:
Tintori, S. C., Sloat, S. A., Rockman, M. V. Rapid isolation of wild nematodes by baermann funnel. Journal of Visualized Experiments. ...