A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans

Abstract

Animal predators can track prey using their keen sense of smell. The bacteriovorous nematode Caenorhabditis elegans employs sensitive olfactory sensory neurons that express vertebrate-like odor receptors to locate bacteria. C. elegans displays odor-related behaviors such as attraction, aversion and adaptation, but the ecological significance of these behaviors is not known. Using a combination of food microbiology and genetics, we elucidate a possible predator-prey relationship between C. elegans and lactic acid bacteria (LAB) in rotting citrus fruit. LAB produces the volatile odor diacetyl as an oxidized by-product of fermentation in the presence of citrate. We show that C. elegans is attracted to LAB when grown on citrate media or Citrus medica L, commonly known as yuzu, a citrus fruit native to East Asia, and this attraction is mediated by the diacetyl odor receptor, ODR-10. We isolated a wild LAB strain and a wild C. elegans-related nematode from rotten yuzu, and demonstrate that the wild nematode was attracted to the diacetyl produced by LAB. These results not only identify an ecological function for a C. elegans olfactory behavior, but contribute to the growing understanding of ecological relationships between the microbial and metazoan worlds.

Figure 1

C. elegans is attracted to lactic acid bacteria grown on media containing citrate. (a) Strategy of study. L. paracasei (LAB) or E. coli strain OP50 is grown on several types of media (media +bacteria) including a rich MRS media (rich), a minimal media (min), minimal media containing glucose (min+glu), minimal media containing citrate (min+citr), minimal media containing both glucose and citrate (min+glu+citr) and nematode growth media (NGM). Media without bacteria (media−bacteria) is used as a control. A 7.5-mm agar plug is taken from each plate and used either for colorimeteric analysis to measure diacetyl production, or attraction behavior assay. In the attraction behavior assay, the +bacteria agar plug is placed on the lid of a 9-cm plate, and the –bacteria agar plug is placed on the opposite side, and C. elegans are allowed to move freely on an agar assay plate (see Materials and Methods). (b) Diacetyl production of L. paracasei grown for 2 days on different types of media. Diacetyl concentration is measured by colorimetric analysis in four separate experiments (Diacetyl standard curves are provided in Supplementary Figure S2). Error bars indicate s.e. (c) C. elegans attraction behavior to diacetyl (1:1000 dilution), E. coli strain OP50 or L. paracasei (LAB) grown for 2 days on rich or citrate media. Error bars indicate s.e. (d) Bacterial colony count (top), diacetyl production (middle) and C. elegans attraction behavior (bottom) to LAB grown on citrate media over 72 h. Colony count is measured as colony forming units (cfu). Diacetyl levels are measured as sample absorbance at 525 nm. Error bars indicate s.e. Significance was calculated by Student's t-test and P-values are indicated.

Figure 2

C. elegans is attracted to diacetyl produced by LAB grown on citrate media. (a) Pre-exposure to the odor diacetyl results in adaptation behavior towards LAB grown on citrate media. Wild-type C. elegans was pre-exposed to 1:5000 dilution diacetyl, 1:5000 dilution benzaldehyde or a control buffer for 80 min, then performed an attraction assay either to diacetyl, LAB grown on citrate media or benzaldehyde. Error bars indicate s.e. Significance was calculated by Student's t-test and P-values are indicated. (b) Attraction was then tested to diacetyl, OP50 on citrate media, or LAB on citrate media with wild-type C. elegans and odr-10(ky32) mutants carrying a mutation in the ODR-10 diacetyl receptor. Error bars indicate s.e. Significance was calculated by Student's t-test and P-values are indicated.

Figure 3

C. elegans is attracted to diacetyl produced by LAB grown on media made from the citrus fruit yuzu. LAB was grown for 2 days on media made from apple, tomato, barley malt, or yuzu, and diacetyl production (a) or attraction behavior of wild-type and odr-10 mutants (b) of an agar plug from the plates was assessed. Diacetyl standard curves (Supplementary Figure S2) were used to calculate diacetyl levels. Error bars indicate s.e. Significance was calculated by Student's t-test and P-values are indicated.

Figure 4

Wild Caenorhabditis strain is attracted to wild LAB strains isolated from yuzu. (a) Attraction behavior of wild-type and odr-10 mutant C. elegans to wild Lactobacillus strain Y091 isolated from rotten yuzu was assessed, when grown on either citrate media or yuzu media. (b) Attraction behavior of wild-type C. elegans and wild C. remanei strain Y01 isolated from rotten yuzu to wild Lactobacillus strain Y091 was assessed. Wild LAB was grown on either citrate or yuzu media. Error bars indicate s.e. Significance was calculated by Student's t-test and P-values are indicated.