Simultaneously, population 2, which gave higher FSC and SSC signals (indicative of larger cell size and higher complexity), was highly abundant in fractions enriched in larger symbionts but nearly absent in gradient fractions enriched in smaller symbionts.

To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation.

Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication.

Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities.

We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.

Here, fueled by the oxidation of reduced sulfur compounds (mainly sulfide), the symbionts fix CO 2 and produce considerable amounts of biomass, which constitutes the hosts source of nutrition ( Cavanaugh et al., 1981; Felbeck et al., 1981; Childress et al., 1991; Goffredi et al., 1997a; Girguis and Childress, 2006 ).

Riftia acquires these nutrients mainly by directly digesting parts of the symbiont population ( Hand, 1987; Boetius and Felbeck, 1995; Hinzke et al., 2019 ).

In addition, the symbionts possibly also actively secrete carbon compounds into the bacteriocytes ( Felbeck and Jarchow, 1998 ).

Thus provided, Riftia grows rapidly and reaches nearly 2 m in body length ( Jones, 1981; Lutz et al., 1994; McClain et al., 2015 ), despite its lack of a digestive system.

This enormous productivity is underpinned by a multitude of symbiosis-specific adaptations of both host and symbiont.

Only small Endoriftia cells and the host bacteriocytes in which they reside appear to undergo cell division, indicating that small and large symbionts belong to a common cell cycle ( Bright et al., 2000; Bright and Sorgo, 2003 ).

Previous microscopy-based studies indicated that small and large Riftia symbionts differ not only with regard to their frequency of cell division, but also with regard to carbon incorporation rates, amount of stored glycogen, and area of sulfur storage vesicles ( Bright et al., 2000; Sorgo et al., 2002; Bright and Sorgo, 2003; Pflugfelder et al., 2005 ).

This suggests that individual cell sizes may indeed have dissimilar metabolic properties.

Unlike previous molecular analyses that studied the uncultured Riftia symbionts metabolic capabilities on the population level in a mixture of all cell sizes (e.g.

Markert et al., 2007; Markert et al., 2011; Gardebrecht et al., 2012 ), precluding comparisons between putative subpopulations, we used a more sensitive approach.

We enriched Endoriftia cells of different sizes by gradient centrifugation of trophosome tissue homogenate and subjected these enriched gradient fractions to separate metaproteomic analyses.

To assess whether these differences might be influenced by naturally occurring variations in energy supply, we conducted our analyses with symbionts from both sulfur-rich and sulfur-depleted trophosomes.

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), transmission electron microscopy (TEM), hybridization chain reaction (HCR)-FISH analyses, and flow cytometry complemented these experiments.

Our results suggest a division of labor between different developmental stages of the symbiont, which leads to remarkable physiological heterogeneity within the symbiont population.

Based on CARD-FISH microscopy, we defined four size ranges ( Figure 1 ): very small symbiont cells (2.0 Figure 1 ).

After the enrichment procedure, small bacterial cells had accumulated in the upper, less dense gradient fractions (top), whereas larger symbionts were enriched in the lower, denser fractions (bottom).

For better visibility, brightness and contrast were adjusted in all images.

Between 300 and 1300 individual cells were measured per filter (average: 590).

B and C ) Symbiont cell size distributions in individual gradient fractions.

While all cell size groups were roughly equally abundant in non-enriched trophosome homogenate (Hom), fraction XS had the highest percentage of symbiont cells in the size range 2.0 m - 3.9 m, fraction S contained most symbiont cells of 3.9 m 5.3 m, etc.

Gradient centrifugation was performed using four biological replicates (n 4) of sulfur-rich trophosome tissue ( B ) and three biological replicates (n 3) of sulfur-depleted trophosome tissue ( C ).

To identify distinguishable bacterial cell populations, we examined Syto9-stained cells in Riftia trophosome homogenate and in gradient fractions from the upper and lower parts of the gradient (enriched in smaller and larger symbionts, respectively) with regard to their light scattering properties.

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