Features governing symbiont persistence in the squid-vibrio association

Abstract

Experimental studies of the interaction between host and symbiont in a maturing symbiotic organ have presented a challenge for most animal-bacterial associations. Advances in the rearing of the host squid Euprymna scolopes have enabled us to explore the relationship between a defect in symbiont light production and late-stage development (e.g. symbiont persistence and tissue morphogenesis) by experimental colonization with specific strains of the symbiont Vibrio fischeri. During the first 4 weeks postinoculation of juvenile squid, the population of wild-type V. fischeri increased 100-fold; in contrast, a strain defective in light production (Δlux) colonized normally the first day, but exhibited an exponential decline to undetectable levels over subsequent weeks. Co-colonization of organs by both strains affected neither the trajectory of colonization by wild type nor the decline of Δlux levels. Uninfected animals retained the ability to be colonized for at least 2 weeks posthatch. However, once colonized by the wild-type strain for 5 days, a subsequent experimentally induced loss of the symbionts could not be followed by a successful recolonization, indicating the host's entry into a refractory state. However, animals colonized by the Δlux before the loss of their symbionts were receptive to recolonization. Analyses of animals colonized with either a wild-type or a Δlux strain revealed slight, if any, differences in the developmental regression of the ciliated light-organ tissues that facilitate the colonization process. Thus, some other feature(s) of the Δlux strain's defect also may be responsible for its inability to persist, and its failure to induce a refractory state in the host.

Keywords: Euprymna scolopes; Vibrio fischeri; luminescence; maintenance; persistence.

Fig. 1

The light organ of E. scolopes, which is located in the center of the mantle cavity, undergoes morphogenesis from the hatchling to the mature condition. Left: The center of the newly hatched juvenile light organ is heart-shaped (subtended by the white dashed line in the confocal micrograph image). This retained portion of the organ will undergo maturation over the first weeks following inoculation with the specific symbiont, V. fischeri. The unique features of the juvenile organ include transparent, superficial fields of ciliated epithelia (golden, with fluorescent label; CellTracker Orange, Molecular Probes, Inc.) that potentiate colonization by the symbiont. These fields begin to regress within days of the initiation of the symbiosis. Right: The bilobed, mature light organ of the adult animal.

Fig. 2

Colonization dynamics of the light organ of E. scolopes by a wild-type (wt; open) or ΔluxCDABEG (Δlux; hatched) strain of V. fischeri over the first 28 days (n = 10–30 squid per time point). The dashed line indicates the limit of detection (~10 CFU/light organ). The median and 25^th to 75^th percentile limits are indicated by the horizontal line and box, respectively, and the bars indicate the range of all values.

Fig. 3

Co-colonization of E. scolopes by a 1:1 mixed inoculum of wild type and ΔluxCDABEG. (a) Mean CFU per light organ of wild-type (open) and ΔluxCDABEG (hatched) portions of the symbiotic population (n = 20 squid per time point). The dashed line is the limit of detection (~10³ CFU per organ) and the standard error of the mean is displayed. (b) Competitive index (CI) of the ΔluxCDABEG strain relative to the wild type in the light organ populations (each circle represents the CI for one animal). The CI was calculated as the ratio of ΔluxCDABEG:wild type; the mean value at each time is indicated by the horizontal line, and the dotted line represents the initial inoculation ratio of 1:1.

Fig. 4

Scanning electron micrographs (SEMs) of the right half of E. scolopes light organs at different stages of maturation over the first 4 weeks post hatch. Left column: nonsymbiotic animals; middle column: Δlux colonized animals; right column: wild type colonized animals. The ciliated epithelial fields, seen on all organs at Day 1, regress at different rates. The white arrows indicate remnants of the ciliated field that are retained longer by Δlux-colonized light organs. Scale bars are 100 μm.