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

In this Review, Visick, Stabb and Ruby describe recent advances in understanding the squid-vibrio symbiosis from the symbiont's perspective.

Reduced gravity, or microgravity, can have a pronounced impact on the physiology of animals, but the effects on their associated microbiomes are not well understood. Here, the impact of modeled microgravity on the shedding of Gram-negative lipopolysaccharides (LPS) by the symbiotic bacterium Vibrio fischeri was examined using high-aspect ratio vessels. LPS from V. fischeri is known to induce developmental apoptosis within its symbiotic tissues, which is accelerated under modeled microgravity conditions. In this study, we provide evidence that exposure to modeled microgravity increases the amount of LPS released by the bacterial symbiont in vitro. The higher rates of shedding under modeled microgravity conditions are associated with increased production of outer-membrane vesicles (OMV), which has been previously correlated to flagellar motility. Mutants of V. fischeri defective in the production and rotation of their flagella show significant decreases in LPS shedding in all treatments, but levels of LPS are higher under modeled microgravity despite loss of motility. Modeled microgravity also appears to affect the outer-membrane integrity of V. fischeri, as cells incubated under modeled microgravity conditions are more susceptible to cell-membrane-disrupting agents. These results suggest that, like their animal hosts, the physiology of symbiotic microbes can be altered under microgravity-like conditions, which may have important implications for host health during spaceflight.