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. 2016 Apr 15;5(4):529-34.
doi: 10.1242/bio.015743.

NGT-3D: A Simple Nematode Cultivation System to Study Caenorhabditis Elegans Biology in 3D

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Free PMC article

NGT-3D: A Simple Nematode Cultivation System to Study Caenorhabditis Elegans Biology in 3D

Tong Young Lee et al. Biol Open. .
Free PMC article

Abstract

The nematodeCaenorhabditiselegansis one of the premier experimental model organisms today. In the laboratory, they display characteristic development, fertility, and behaviors in a two dimensional habitat. In nature, however,C. elegansis found in three dimensional environments such as rotting fruit. To investigate the biology ofC. elegansin a 3D controlled environment we designed a nematode cultivation habitat which we term the nematode growth tube or NGT-3D. NGT-3D allows for the growth of both nematodes and the bacteria they consume. Worms show comparable rates of growth, reproduction and lifespan when bacterial colonies in the 3D matrix are abundant. However, when bacteria are sparse, growth and brood size fail to reach levels observed in standard 2D plates. Using NGT-3D we observe drastic deficits in fertility in a sensory mutant in 3D compared to 2D, and this defect was likely due to an inability to locate bacteria. Overall, NGT-3D will sharpen our understanding of nematode biology and allow scientists to investigate questions of nematode ecology and evolutionary fitness in the laboratory.

Keywords: C. elegans; Reproduction; Three dimensions.

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Design and strategy for the NGT-3D experiment. (A) Illustration of NGT-3D production. (B) Experimental strategy and method to assess reproductive fitness by relative brood size.
Fig. 2.
Fig. 2.
Fertility, growth and lifespan of wild-type C. elegans on NGM plates and NGT-3D. (A) Relative brood size of wild-type worms in NGT-3D with fewer than 60 OP50 colonies, greater or equal to 60 colonies, or on 2D NGM plates. Error bars indicate standard error. (B) Scatter plot of worm populations in the L3, L4 and adult developmental stages and total worms against the number of colonies in NGT-3D. Logarithmic regressions are plotted as dotted lines and the correlation coefficient R2 is calculated. Significance of regression is indicated. (C) Percent of F1 generation worms in the L3, L4 or adult developmental stage in NGT-3D with fewer than 60 OP50 colonies, greater than or equal to 60 colonies, or on 2D NGM plates. (D) Scatter plot of the percent of worms in the L3 and adult developmental stages and total worms against the number of colonies in NGT-3D. Logarithmic regressions are plotted as dotted lines and the correlation coefficient R2 is calculated. Significance of regression is indicated. (E) Survival curve of wild-type C. elegans in NGT-3D or NGM plates. N.S. indicates not significantly different calculated by log-rank test.
Fig. 3.
Fig. 3.
Animals defective in osm-6 show compromised brood size in NGT-3D. (A) Percent difference between relative brood size in NGT-3D with more than 40 OP50 colonies and NGM plates is shown for N2 wild-type and several types of mutants. Sample size: N2, n=22; daf-2, n=8; sek-1, n=6, eat-2, n=5; tax-2, n=6; osm-6, n=5; osm-6; OSM-6::GFP, n=6. (B,C) Relative brood size was determined for N2 and osm-6 mutants and osm-6;OSM-6::GFP rescue strain on OP50 dot plates (B) and OP50 patch plates (C) with varying numbers of bacterial colonies. See Materials and methods for explanation of the types of plates. Sample size n=5 for all. Error bars indicate standard error. Significance is calculated by Student's t-test. *P<0.05; **P<0.01; ***P<0.001.

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