Chemical basis of microbiome preference in the nematode C. elegans

Animals are exposed to many microbes in their environment, some of which have been shown to colonize various tissues including the intestine. The composition of the intestinal microbiota affects many aspects of the host’s physiology and health. Despite this, very little is known about whether host behavior contributes to the colonization. We approach this question in the nematode C. elegans, which feeds on bacteria and also harbors an intestinal microbiome. We examined the behavior of C. elegans towards CeMbio, a simplified microbiome consisting of twelve strains that represent the bacteria found in the animal’s natural environment. We observed that C. elegans raised on E. coli shows a strong preference for three members of CeMbio (Lelliottia amnigena JUb66, Enterobacter hormaechei CEent1, and Pantoea nemavictus BIGb0393) compared to E. coli. Previously, these three bacterial strains have been shown to support faster C. elegans development time than E. coli OP50 and are low colonizers compared to eight other members of CeMbio. We then used gas chromatography coupled to mass spectrometry to identify that these three bacteria release isoamyl alcohol, a previously described C. elegans chemoattractant. We suggest that C. elegans seeks bacteria that release isoamyl alcohol and support faster growth.

support a shorter development time than five of the CeMbio strains and E. coli OP50, and are low colonizers of the intestine and so are likely nutritious food sources.We then used gas chromatography coupled with mass spectrometry (GC-MS) to identify the major volatile chemicals released by the bacterial isolates and found that all three attractive isolates released isoamyl alcohol, a well-studied chemoattractant [13][14][15] .

Olfactory preference behavior of C. elegans for CeMbio
To determine the preference of C. elegans for CeMbio bacterial species based only on volatile chemicals, we used a bacterial odor choice assay in which worms raised on E. coli OP50 are given a choice between the CeMbio bacteria and E. coli OP50, placed on the petri dish lid 16 .In this assay, where C. elegans only uses volatile chemical cues released by the bacteria to discriminate among the two bacterial species, C. elegans showed a significant preference for three bacterial strains, Lelliottia amnigena JUb66, Enterobacter hormaechei CEent1, and Pantoea nemavictus BIGb0393 over E. coli OP50 (Fig. 1).To our knowledge, this is the first time the attractiveness of JUb66 and CEent1 has been demonstrated.It has previously been shown that Pantoea nemavictus BIGb0393 and other Pantoea sp. are attractive to C. elegans over E. coli OP50 in bacterial choice assays 17 .In addition, consumption of Enterobacter hormaechei CEent1 has been shown to provide immune-protective effects in C. elegans 6 .Two of the preferred strains (JUb66 and CEent1) represent the most abundant family of bacteria, Enterobacteriaceae, found in the natural environment of C. elegans 6 .Until recently Pantoea BIGb0393 was thought to belong to the Enterobacteriaceae family, but now the genus Pantoea is classified as part of the Erwiniaceae family which is closely related to the Enterobacteriaceae family 18 .
C. elegans showed no preference in choice assays between the remaining nine CeMbio strains and E. coli OP50 (Fig. 1).The choice index for these bacterial strains vs. E. coli OP50 did not differ significantly from the choice index between two patches of E. coli OP50.We hypothesize that this is because neither the CeMbio strain or E. coli OP50 are releasing sufficient amounts of attractive or repulsive volatile chemicals under these low nutrient assay conditions to produce a consistent preference.Therefore, the worms are likely not using the volatile cues

Choice Index
CeMbio bacterial strain

Discussion
We examined the odor preference of C. elegans for different bacterial species that comprise the simplified representative microbiome of C. elegans, CeMbio.We found that C. elegans showed a significant preference for three strains (Lelliottia JUb66, Enterobacter CEent1, and Pantoea BIGb0393) over E. coli OP50 and that all three of these strains released isoamyl alcohol, a known C. elegans chemoattractant.
Our study shows that C. elegans prefers three of the 12 CeMbio bacteria compared to the standard laboratory food source E. coli OP50 strain.Previous studies examining the individual strains that comprise CeMbio identified several characteristics of these bacteria 6 .First, we compared bacterial preference to developmental time.Nine CeMbio strains resulted in a faster development time compared to E. coli OP50, while two strains (Sphingobacterium BIGb0170 and Chryseobacterium JUb44) resulted in a slower time and one strain had a similar time to E. coli OP50.C. elegans showed a strong preference for Pantoea nemavictus BIGb0393 which resulted in the second greatest percentage of adults at 52 h post L1 6 .Lelliottia amnigena JUb66 supported a developmental time above the median for all CeMbio strains and Enterobacter hormaechei CEent1 supported a developmental time just below the median.C. elegans did not show a preference for several of the species that supported a growth time shorter than the median (Pseudomonas lurida MYb11, Sphingomonas molluscorum JUb134, Pseudomonas berkeleyensis MSPm1, Stenotrophomonas JUb19, Comamonas BIGb0172).These results suggest that based on volatile cues C. elegans prefers some, but not all, bacterial strains that confer a faster growth time.Overall, there is a low correlation between development time and bacteria choice index in this study (linear regression r 2 = 0.23).This is consistent with previous study of bacterial strains found in the natural environment of C. elegans, but not part of CeMbio, where a strong correlation (r 2 = 0.52) was not found between bacterial preference and development time 16 .
We next examined the correlation between behavioral preference for bacteria and the level of bacterial colonization of the intestine.Dirksen   www.nature.com/scientificreports/individually the intestine after 72 and 120 h into three groups: low colonizers, medium colonizers, and high colonizers.It is of note that the bacterial strains for which C. elegans showed the highest preference were all low colonizing strains (Lelliottia amnigena JUb66, Enterobacter hormaechei CEent1, and Pantoea nemavictus BIGb0393).The other low colonizing bacteria was Acinetobacter guillouiae MYb10 for which C. elegans showed no preference over E. coli OP50.When C. elegans is cultured on the community of all twelve strains of CeMbio, these preferred strains are also low colonizers of C. elegans intestine 6 .Therefore, based on olfactory cues, C. elegans prefers low colonizing bacterial strains.Low colonizing bacterial strains may be preferred because they provide more nutrition to C. elegans.
In addition, Dirksen et al. analyzed the predicted number of metabolic pathways present each bacterial strain from genomic data.The three preferred strains have the three highest number of metabolic pathways of all the CeMbio strains.Enterobacter CEent1 has the highest number of metabolic pathways, 386, while Chryseobacterium JUb44 has the fewest, 186 pathways.The high number of metabolic pathways likely indicates that the preferred bacterial strains can be metabolically active in a wide variety of environments which provide different nutrient sources for growth.Specifically, a well-studied metabolic pathway that produces isoamyl alcohol as a byproduct is the catabolism of leucine by the Ehrlich pathway in Saccharomyces cerevisiae 23 .Based on protein predictions from genomic sequence data 6 , the enzymes that comprise this pathway are not present in the preferred bacterial strains.Further research is required to determine the metabolic pathway or pathways in each of the preferred strains that result in the release of isoamyl alcohol.
In addition to the preferred strains having the highest number of metabolic pathways, two of the attractive strains, Lelliottia JUb66 and Enterobacter CEent1, belong to the bacterial taxa that is found most often in the  www.nature.com/scientificreports/natural habitat of C. elegans.Each CeMbio strain was chosen as a representative of the most abundant bacterial taxa found in different natural environments of C. elegans, and Lelliottia JUb66 and Enterobacter CEent1, represent the most abundant bacterial taxa of all the CeMbio strains (operational taxonomic unit, or OTU1) 6 .A likely hypothesis is that C. elegans likely coexists with this bacterial taxon most often because they are most attracted to them.In the future, it would be interesting to examine other bacterial isolates belonging to this taxon to determine if they are also attractive.
Taken together, in this study, C. elegans showed olfactory preference for three CeMbio strains (Lelliottia JUb66, Enterobacter CEent1, and Pantoea BIGb0393).These strains are likely nutritional food sources, low colonizers of the intestines, and have the highest number of metabolic pathways.In addition, two of the attractive strains, Lelliottia JUb66 and Enterobacter CEent1 represent the most abundant bacterial taxa of all CeMbio strains.C. elegans is found naturally in bacterial-rich environments, particularly rotting fruit and compost heaps, shows innate preferences for different species of bacteria, and can detect and recognize volatile chemicals released from bacteria 12,24 .However, only recently, the chemical cues released by bacteria that C. elegans prefers have begun to be defined 16,19-2125-28 .In this study and a previous study, isoamyl alcohol was found to be released by preferred bacterial strains.In a previous study, bacteria found in the natural environment but not part of CeMbio, four of the six attractive isolates (Alcaligenes JUb4, Providencia JUb5, Providencia JUb39, and Flavobacteria JUb43) were also found to release isoamyl alcohol 16 .The robust attractive response of C. elegans to isoamyl alcohol from undiluted to 10 −4 dilution is well-studied [13][14][15] .The next question is to begin to understand the basis of preference or why C. elegans is attracted to some volatile chemicals released by bacteria and not by others.Perhaps, isoamyl alcohol often indicates an actively growing bacteria and an actively growing bacteria is likely to be a nutritious food source for C. elegans.
In the future, it would be interesting to examine other factors that affect C. elegans olfactory preferences for bacterial strains.Culturing bacteria in different defined media with different nutrient sources would likely result in utilization of different bacterial metabolic pathways and volatile profiles which may change olfactory preferences of C. elegans.For example, in this study C. elegans showed no preference for Pseudomonas MSPm1 or Pseudomonas MYb11.However, in another study C. elegans was found to be attracted to MSPm1 over E. coli HB101 and to be repulsed by MYb11 29 .In this other study, bacteria were resuspended in LB rather than PBS as in the current study.The LB provided more nutrients so that the bacteria likely released more and/or different volatile chemicals that mediated attraction or repulsion by C. elegans.
In addition to examining the effects of different nutrient sources to support bacterial growth, it would be interesting to examine olfactory responses to different mixtures of CeMbio strains which may result in different volatile chemicals being produced because the bacteria can use metabolites produced by each other to grow.For example, co-cultures of MYb71 and MYb11 produce different compounds compared with individual cultures of the two species 30 .The attractiveness and volatile profile of this combination of bacterial strains as well as other mixtures of CeMbio strains can be examined.
Overall, a goal of future work would be to define the volatile cues that attract C. elegans to bacterial strains under different bacterial growth conditions.The next step is to determine if there are consistent characteristics of the preferred bacteria that these cues signify, such as an indication of actively growing bacteria.This work contributes to a foundation for future work to understand better how C. elegans behavior affects interactions with its microbiome.

Bacterial odor choice assay
The bacterial odor choice assay which measures olfactory preference for bacteria was modified from Worthy et al. in order to follow the protocol used in Dirksen et al. for preparing CeMbio bacteria.Briefly, CeMbio bacterial strains and E. coli OP50 were grown overnight (except JUb134 was grown for 48 h because it grows more slowly than other CeMbio strains) in Luria Broth (LB) at 25 °C.25 μL of each bacterial suspension (OD 600 = 10) in PBS was spotted onto NGM plates and incubated for 24 h at room temperature.Then each agar square containing 25 μL bacteria patch was extracted using a sterile metal spatula.An NGM agar square with CeMbio bacterial isolate and an NGM agar square with E. coli OP50 were placed on opposite sides of a petri dish lid. 1 μL of 1 M sodium azide was pipetted on NGM agar directly below bacterial patch on lid to immobilize worms.Adult animals were washed three times in S-basal buffer, 50 to 250 were placed in the center of the NGM plate, equidistant from the two bacterial patches.Animals were allowed to move freely for 1 h and then were counted.The bacterial choice index is the number of worms on the CeMbio strain minus the number of worms on E. coli OP50 divided by the number of worms underneath both bacterial patches.

Gas chromatography-mass spectrometry
Bacteria were prepared for GC-MS analysis in a similar method as described previously 16 .Bacteria were grown overnight in LB at 25 °C, centrifuged, and then resuspended in PBS at an OD 600 = 10.Two NGM plates were prepared each with 9 spots of 25 μL of bacterial suspension.For the controls, 25 μL of PBS without bacteria was spotted on NGM plate.Plates were incubated for 1 h at 20 °C.Then 18 squares of the NGM agar with 25 μL bacteria suspension were placed in a GC-MS glass vial for 24 h at 20 °C.Headspace samples were collected using Markes PDMS-coated HiSorb probes and were analyzed by Thermal Desorption (TD) GC-MS using the Agilent 6890 GC System equipped with a Markes Unity II Thermal Desorption System on the GC inlet, a Restek, Rtx-5 column, and Agilent 5973 Mass Selective Detector.The thermal desorption sampling method was used rather than a solid-phase microextraction (SPME) fiber to increase sensitivity 31 .The temperature program was hold 8 min at 35 °C, increased to 130 °C at a rate of 10 °C/min, hold 5 min at 130 °C then increased to 300 °C at a rate of 15 °C/min, and hold at 300 °C for 1 min.MS ranged from m/z 30 to 550 in full scan mode.VOCs were identified with the NIST 11 (National Institute of Standards and Technology) mass spectral library and pure chemical standards run following the same parameters as for bacterial samples for all chemicals except for cycloundecene and 4-undecene which could not be obtained.Samples were prepared for analysis in duplicate on different days from a single stock of each bacterium and PBS control samples were run immediately before or after each bacterial sample.

Chemotaxis assays
Chemotaxis assays were performed using 10 cm square chemotaxis plates as described 32 .In brief, assay agar was 2% agar, 1 mM MgSO 4 , 1 mM CaCl 2 , 5 mM phosphate buffer [pH 6.0].Chemical dilutions were in ethanol at the concentrations indicated in figure legends. 2 μL of diluted chemical was pipetted on one side of the plate, 2 μL of ethanol on the other side, and 2 μL of 1 M sodium azide on both sides to anaesthetize animals that reached odor or ethanol sources.Adult animals were washed two times in S-basal buffer and one time in water, 50-200 animals were placed at the center of chemotaxis plate, plate was covered with lid, and the distribution of animals counted after 1 h.

Statistical analysis
Means represent data pooled from assays run on at least two different days with at least 6 replicates.Error bars in all figures are standard error of means.The data were analyzed using statistics described in figure legends with GraphPad Prism v9.5 for Mac (GraphPad Software, San Diego California USA) or Microsoft Excel.
et al. categorized the CeMbio bacterial strains for their ability to colonize

Figure 2 .
Figure 2. Gas chromatography-mass spectrometry of headspace of CeMbio bacterial strains.Overnight liquid cultures of bacteria were spotted on NGM agar plates (OD 600 = 10) and incubated for 1 h, then NGM agar squares with bacterial suspension were placed inside a GC-MS glass vial for 24 h.One asterisk indicates "probe peaks, " i.e. volatile cyclic siloxanes released by the HiSorb probe.Peaks were identified tentatively with NIST 11 (National Institute of Standards and Technology) mass spectral library and confirmed with known standards except for cycloundecene and 4-undecene which could not be obtained.All bacterial samples were analyzed two or more times on different days.(A) Bacterial strains which volatile organic compounds were detected.Preferred strains are shown in pink; non-preferred strains are shown in blue.(B) Bacterial strains where no volatile organic compounds were detected.

Figure 3 .
Figure 3. Chemotaxis to volatile organic compound released by attractive CeMbio strains.(A) 1-methoxy-3-methylbutane (shown to be released by Lelliottia JUb66) and diluted in ethanol.(B) 1-nonene (shown to be released by Pseudomonas lurida MYb11) and diluted in ethanol.n ≥ 6 assays conducted on at least two different days.Error bars represent SEM.