Achieving a multi-strain symbiosis: strain behavior and infection dynamics

Abstract

Strain diversity, while now recognized as a key driver underlying partner dynamics in symbioses, is usually difficult to experimentally manipulate and image in hosts with complex microbiota. To address this problem, we have used the luminous marine bacterium Vibrio fischeri, which establishes a symbiosis within the crypts of the nascent light organ of the squid Euprymna scolopes. Competition assays in newly hatched juvenile squid have shown that symbiotic V. fischeri are either niche-sharing "S strains", which share the light organ when co-inoculated with other S strains, or niche-dominant "D strains", which are typically found alone in the light organ after a co-colonization. To understand this D strain advantage, we determined the minimum time that different V. fischeri strains needed to initiate colonization and used confocal microscopy to localize the symbionts along their infection track. Further, we determined whether symbiont-induced host morphogenic events also occurred earlier during a D strain colonization. We conclude that D strains colonized more quickly than S strains. Nevertheless, light-organ populations in field-caught adult squid often contain both D and S strains. We determined experimentally that this symbiont population heterogeneity might be achieved in nature by a serial encounter of different strains in the environment.

Fig. 1

Minimum exposure time needed for V. fischeri strains to initiate a successful colonization. Squids were exposed to each bacterial strain for different periods of time, then rinsed, and colonized animals identified by their production of bioluminescence after 24 h. Strains in bold are D strains, whereas the others are S strains. The X-axis indicates the percentage of squid colonized after a given length of exposure. Strains are ranked (top to bottom) based on the minimum amount of exposure needed to colonize 50% of the animals. For each condition, between 25 and 31 animals were measured in three replicates. Error bars indicate 95% confidence intervals

Fig. 2

Strain localization during colonization. a Confocal image of a light organ being colonized by GFP-labeled V. fischeri (green); tissue nuclei (blue) were stained with TOTO-3. Some bacteria were in transit (orange dashed area) and some were in the crypts (blue dashed area). One crypt is visualized in this optical section, but all crypts were examined for the analysis. b Schematic representation of one side of the squid light organ. V. fischeri strains enter through the pores and, after transit, reach the crypts where they grow and luminesce. c Using confocal microscopy, we identified where GFP-labeled bacterial cells were located in the light organ at different times after inoculation. For each light organ, we determined whether there was no evidence the bacteria had begun migrating into the tissues (white) or, when bacteria were present, whether they were still in transit (orange) or had reached the crypts (blue) and begun growing. Between 19 and 23 animals were analyzed in two replicates. The graph represents the mean percentages for each result, and the error bars the 95% confidence interval

Fig. 3

Induction of symbiont-induced regression in the host light organ. a Squids were colonized by one dominant (D₁ or D₂) or sharing (S₁ or S₂) strain for 18 h (aposymbiotic, or apo = non-colonized animals), after which the extent of regression of the organ’s appendages was determined for each condition by measuring the mean longitudinal cross-sectional area of the anterior appendage. Each dot corresponds to an individual squid. Between 29 and 35 squids were analyzed in two to three replicates. Means with standard deviations are presented. Differences between conditions were assessed by a Dunn’s test; letters above the columns indicate significantly different means (p < 0.05). b Squids were colonized by a single strain for 18 or 41 h, and observed by scanning electron microscopy to assess regression of the anterior (aa) and posterior (pa) appendages; bars = 60 mm. After colonization by strain D₂, but not S₂, both appendages are almost totally regressed by 41 h

Fig. 4

State of colonization of juvenile squid encountering two strains presented sequentially. Squids were inoculated with strain D₂ or S₂ at time zero (t₀), and strains D₁ or S₁ were added into the surrounding water at different times (t, in hours; X-axis). The percentage of squids either co-colonized (gray), or colonized only by the strain added either initially (white) or later (black), were determined. Between 29 and 38 animals were analyzed in three replicates. The graph represents the mean percentages for each result, and the error bars the 95% confidence interval

Fig. 5

Strain localization in the crypts of the light organ. Squids were inoculated with one of four different strain combinations: (i) two S strains, (ii) two D strains, or (iii) an S strain, with a D strain added either simultaneously, or (iv) after a 3 h delay. (a) After 48 h, squids were fixed, and their light organs observed under a confocal microscope to determine the percentage of their 6 colonized crypts (gray outline) that were singly colonized by a GFP- (G) or an RFP- (R) labeled strain or were co-colonized by both of the inoculated strains (GR). (b) For each treatment, 53 or 54 animals were analyzed in four replicates, corresponding to between 187 and 217 colonized crypts observed per treatment. The bar graph represents the mean percentage of each outcome, and the error bars the 95% confidence interval. The crypts analyzed were either co-colonized (gray) by both of the inoculating strains, or were only singly colonized by one of the pair of strains (white) or the other (black)

Fig. 6

a The composition of symbiont populations in squid inoculated with three strains. Animals were first exposed to an inoculum of two S strains (S₁ and S₂; black and white ovals); after 3 h, strain D₂ (red ovals) was added. After 72 h, we determined the percentage of colonized squid that had one of the 6 possible combinations of the three inoculated strains. One hundred squids were analyzed in three replicates. The graph represents the mean percentage, and the error bars the 95% confidence intervals. b Model for the colonization of a light organ’s crypts with a mixture of S and D strains. Two scenarios are given (left and right panels) indicating how the crypts might become colonized, depending on when the host encounters the two kinds of strains. Each scenario is illustrated using three crypts that depict different possible trajectories for the progression of colonization: (i) when all three strains (S₁, S₂ and D₂) are added at the same time (left panel), the D strain will migrate more rapidly than the S strains, which cannot reach the deep crypts before they have been colonized, and the bottleneck has been closed by the host in response to the presence of bacteria in the crypt [30]; (ii) when there is a sequential encounter of strains (right panel), S strains have the opportunity to migrate into and colonize at least some crypts, while D strains encountered 3 h later (red arrow) can only colonize any remaining empty crypts whose bottleneck has not been closed