Associations between animals and microbes affect not only the immediate tissues where they occur, but also the entire host. Metabolomics, the study of small biomolecules generated during metabolic processes, provides a window into how mutualistic interactions shape host biochemistry. The Hawaiian bobtail squid, Euprymna scolopes, is amenable to metabolomic studies of symbiosis because the host can be reared with or without its species-specific symbiont, Vibrio fischeri. In addition, unlike many invertebrates, the host squid has a closed circulatory system. This feature allows a direct sampling of the refined collection of metabolites circulating through the body, a focused approach that has been highly successful with mammals. Here, we show that rearing E. scolopes without its natural symbiont significantly affected one-quarter of the more than 100 hemolymph metabolites defined by gas chromatography mass spectrometry analysis. Furthermore, as in mammals, which harbor complex consortia of bacterial symbionts, the metabolite signature oscillated on symbiont-driven daily rhythms and was dependent on the sex of the host. Thus, our results provide evidence that the population of even a single symbiont species can influence host hemolymph biochemistry as a function of symbiotic state, host sex and circadian rhythm.

Sheet 1: Relative expression values of ssrA from bacteria cells fraction or OMV fractions. Cells grown in three different media: a tryptone-based medium (LBS) or LBS with the addition of either glycerol (32.6 mM) or GlcNAc (10 mM). S1A Fig. Sheet 2: OD600 values over 24 h of bacteria growth in tryptone-based medium (LBS). S1A Fig. Sheet 3: OD600 values over 24 h of bacteria growth in minimum medium. S1A Fig. Sheet 4: Motility in soft agar of WT or DeltassrA cells measured as the diameter of the outer migration ring at 3 and 7 h post inoculation. S2B Fig. Sheet 5: Respiration rates of WT, DeltassrA, DeltassrA + ssrA, and a nonluminescent lux-deletion mutant (Deltalux) normalized to OD600. Fig 2C. Sheet 6: RCI between WT and DeltassrA in co-inoculated light organs after 24, 48, and 72 h. S2D Fig. Sheet 7: Relative expression values of ssrA and smpB. S2E Fig. GlcNAc, N-acetyl-glucosamine; LBS, Luria-Bertani salt medium; OD600, optical density at 600 nm; OMV, outer membrane vesicle; RCI, relative competitive index; WT, wild type. (XLSX) Copyright: CC BY 4.0

Sheet 1: Survival proportions of juvenile squid colonized by either WT, DeltassrA, DeltassrA + ssrA, or DeltasmpB. Fig 4B, S6B Fig. Sheet 2: Dry weight of juvenile squid immediately after hatching ("Hatch") or at 4 d post hatching when kept APO or colonized with WT, DeltassrA, or a nonluminescent mutant (Deltalux) strain. Fig 4C. Sheet 3: Quantification of internal yolk-sac area of juvenile squid immediately after hatching ("Hatch") or at 2 d post hatching colonized with WT or DeltassrA. Fig 4D. APO, aposymbiotic; WT, wild type. (XLSX) Copyright: CC BY 4.0

Sheet 1: Counts in OMV and hemolymph samples. Sheet 2: Numerical values for Fig 1B. Sheet 3: Differential-expression analysis (Fig 1C). OMV, outer membrane vesicle; RNA-seq, RNA sequencing. (XLSX) Copyright: CC BY 4.0

. Copyright: CC BY 4.0

Sheet 1: Relative expression values of C3. Fig 5B. Sheet 1: Relative expression values of RIG-I. Fig 5B. C3, complement protein 3; RIG-I, retinoic-acid inducible gene-I. (XLSX) Copyright: CC BY 4.0

Sheet 1: Survival proportion of juvenile squid that were either single-colonized by WT or DeltassrA, or co-colonized at a 1:1 inoculum ratio with both WT and DeltassrA (n = 60). S6C Fig. Sheet 2: Respiration rates of newly hatched squid ("Hatch"), or of animals after 24 h, that were either maintained APO or colonized by WT, DeltassrA, or Deltalux strains. S6D Fig. Sheet 3: Internal yolk-sac area values, 2 d post colonization with WT, DeltassrA, its complement (DeltassrA + ssrA), the nonluminescent mutant (Deltalux), or DeltasmpB strains. S6E Fig. Sheet 4: Quantification of laccase-3 signal by HCR using relative fluorescence intensity of a Z-series image of the light organ. S7A Fig. Sheet 5: Quantification of laccase-3 presence by HCR fluorescence signal intensity from a Z-series image of light organs, 3 h after incubation with WT or DeltassrA OMVs. S7B Fig. APO, aposymbiotic; HCR, hybridization chain reaction; OMV, outer membrane vesicle; WT, wild type. (XLSX) Copyright: CC BY 4.0

Sheet 1: CFU per squid. Fig 2A. Sheet 2: Number of hemocytes trafficking into the light-organ appendages after 16 and 18 h post colonization. Fig 2B. Sheet 3: Number of hemocytes trafficking into the light-organ appendages after 3 h inoculation with WT or DeltassrA OMVs. Fig 2C. Sheet 4: Number of apoptotic nuclei per appendage. Fig 2D. Sheet 5: RLU per CFU of symbionts either within the light organ, or within a homogenate of the light organ, of a 24-h juvenile. Fig 2E. CFU, colony-forming units; OMV, outer membrane vesicle; RLU, relative light units. (XLSX) Copyright: CC BY 4.0