Ciliated epithelia are key elements in the recruitment of bacterial partners in the squid-vibrio symbiosis

Abstract

The Hawaiian bobtail squid, Euprymna scolopes, harvests its luminous symbiont, Vibrio fischeri, from the surrounding seawater within hours of hatching. During embryogenesis, the host animal develops a nascent light organ with ciliated fields on each lateral surface. We hypothesized that these fields function to increase the efficiency of symbiont colonization of host tissues. Within minutes of hatching from the egg, the host's ciliated fields shed copious amounts of mucus in a non-specific response to bacterial surface molecules, specifically peptidoglycan (PGN), from the bacterioplankton in the surrounding seawater. Experimental manipulation of the system provided evidence that nitric oxide in the mucus drives an increase in ciliary beat frequency (CBF), and exposure to even small numbers of V. fischeri cells for short periods resulted in an additional increase in CBF. These results indicate that the light-organ ciliated fields respond specifically, sensitively, and rapidly, to the presence of nonspecific PGN as well as symbiont cells in the ambient seawater. Notably, the study provides the first evidence that this induction of an increase in CBF occurs as part of a thus far undiscovered initial phase in colonization of the squid host by its symbiont, i.e., host recognition of V. fischeri cues in the environment within minutes. Using a biophysics-based mathematical analysis, we showed that this rapid induction of increased CBF, while accelerating bacterial advection, is unlikely to be signaled by V. fischeri cells interacting directly with the organ surface. These overall changes in CBF were shown to significantly impact the efficiency of V. fischeri colonization of the host organ. Further, once V. fischeri has fully colonized the host tissues, i.e., about 12-24 h after initial host-symbiont interactions, the symbionts drove an attenuation of mucus shedding from the ciliated fields, concomitant with an attenuation of the CBF. Taken together, these findings offer a window into the very first interactions of ciliated surfaces with their coevolved microbial partners.

Keywords: Euprymna scolopes; Vibrio fischeri; ciliary beat frequency; ciliated fields; light organ; symbiont harvesting.

FIGURE 1

The light organ of Euprymna scolopes, and relevant early post-hatch events in the establishment of symbiosis. (A) The position of the organ in the body cavity. Left, a light micrograph of a juvenile E. scolopes, showing the location of ink-sac-associated light organ within the body cavity (orange box). Right, a diagram of a hatchling squid with a portion of the mantle removed to reveal a confocal micrograph (inset) of the internal light organ with its lateral ciliated fields (orange arrows). (B) Early development of the light organ under natural conditions post symbiont colonization (left) and the timeline of the associated events (right). Upper left—at hatching, each exterior lateral surface is covered with two distinct populations of cilia: (i) longer metachronally-beating cilia (lc; darker blue areas) along the outer regions of the anterior (ap) and posterior (pa) appendages, and along the medial ciliated ridge (cr); and, (ii) shorter randomly beating cilia (sc) (lighter blue areas), which cover the inner surfaces of the appendages, and surround the pores (p) into which the symbionts migrate. Following recruitment to a stagnation zone above the pores, V. fischeri cells will migrate into the pores and down a migration path into three blind-ended crypts (1, 2, 3) on each side of the organ, where they will proliferate and populate these spaces. Lower left—by ∼4d, colonizing symbionts (green) induce a developmental program that results in a gradual loss of the superficial ciliated fields.

FIGURE 2

Analyses of the ciliated field that potentiates host colonization by the bacterial symbiont. (A) A still image from a high-speed video (×40, DIC, 1,000 frames/sec) of one half of the hatchling squid light organ, showing the long (lc) and short (sc) cilia; p, pore. (A′) Left, a MATLAB algorithm detects areas of motion (white); middle and right, speed of ciliary beat, calculated using fast Fourier transform (FFT) of each window, at 30 min post hatching of the host animal into either artificial (ASW) or natural (NSW) seawater. (B) Kymographs showing the changes in gray values over time of the ciliary beat in the long (metachronally beating) and short (randomly beating) cilia, respectively; t, time; x/y = pixel location. (C) Periodic changes in gray value over distance (pixels), which represents time corresponding to the beat pattern of the metachronally beating, long cilia. Filtering of high and low frequency noise and artifacts, followed by FFT and averaging to extract a spectrum of frequencies (Hz) at this location. (D) Derived ciliary beat frequency (CBF) of animals exposed for 30 min post hatching to either ASW or NSW; CBF (Hz), ciliary beat frequency in Hertz; (n = 10); ****, p < 0.0001.

FIGURE 3

The influence on CBF of peptidoglycan (PGN)-induced mucus shedding in the hatchling light organ. (A) Confocal images of relevant post-hatch events. Top, within minutes, PGN shed from environmental bacteria has induced a non-specific release of copious mucus from the superficial ciliated fields on either side of the organ; in this image the labeling of the cilia is obscured by the mucus, except at the pores (p). aa, anterior appendage; hg, hindgut; pa, posterior appendage. Lower left—region of dashed box in top image rotated around its axes to show the mucus as it typically resides above the ciliated field. Lower right—NO (red) occurs in vesicles in the mucus. (B) Responses of CBF to the presence of PGN, and manipulation of the NO cue present in the mucus. (See Materials and Methods for conditions of exposure.) *p < 0.05; **p < 0.01; ***p < 0.001; ****p > 0.0001; ns, not significant. (C) The effect of PGN-induced mucus shedding on host colonization. Animals were exposed to 500–800 V. fischeri/ml of seawater with either no PGN or 200 µg PGN/ml added for 2 h, and the luminescence was measured after 24 h; all animals that subsequently emitted luminescent were considered colonized; five replicates were made from different clutches of host eggs; each replicate had 10–14 animals/condition. The summary statistics of these two experimental conditions are a mean of (i) 17 (±19.7) without added PGN, and (ii) 60 (±25.5) with added PGN. In a paired plot of these two conditions, the dashed lines between points connect the two replicates, and the boxes are the interquartile ranges for each condition. Both the summary and pairing statistics suggest that further analysis would be informative. Nevertheless, to determine the effect of PGN-induced mucus shedding, we used a paired-sample t-test, with the null and the alternative hypotheses corresponding to the two conditions being either not different or statistically different, respectively. The t- test’s p-value (0.0058) indicates that the two conditions have significantly different outcomes.

FIGURE 4

Ciliary beat frequency (CBF) measurements of hatchling light organs show exquisite sensitivity to Vibrio fischeri. Animals were incubated with various concentrations of V. fischeri (Vf) in natural seawater containing ∼10⁶ CFU/ml of other marine bacteria. As controls, animals were also exposed to only natural seawater with no additions (NA) or to addition of the same volume of cell-free 0.22 µm-filtered spent LBS-medium as presented with Vf cells (Medium). A one-way ANOVA and Tukey’s post hoc test were used (F_{6, 79} = 6.251, p < 0.0001). Animals (n) per treatment: NA, n = 12; medium control, n = 10; medium with 500 CFU/ml for 30 min, n = 13; 5,000 CFU/ml 5 min, n = 12; 5000 CFU/ml 15 min, n = 11; 5,000 CFU/ml 30 min, n = 17; 5,000 CFU/ml, 60 min, n = 11.

FIGURE 5

Ciliary beat frequency (CBF) measurements of hatchling light organs show no effect of symbiont viability or certain surface features or behaviors. (A) Conditions and features that show no difference in CBF and pattern of CBF in comparison with exposure to viable wild-type V. fischeri. In all conditions, animals were exposed to 5,000 V. fischeri CFU/ml for 30 min and then CBF was measured. For statistical analyses one-way ANOVA and Dunnett’s multiple comparisons test were used (F_{5, 67} = 4.86, p = 0.335). Animals (n) per treatment: viable, n = 19; heat-killed, n = 10; hyperswimmer, n = 11; unflagellated immotile, n = 11; flagellated immotile, n = 8; no O-antigen, n = 11. For the pilus mutant, a one-way ANOVA and Tukey’s post hoc test were also used (F_3,47 = 3.428, p = 0.0245), n = 11; (B,B′) While exposure to the pilus mutant showed no overall difference in CBF (B), an example of a single animal; see Figure 2 for the heat-map patterns for wild-type V. fischeri, the variance of CBF measurements between individual light organs was significantly higher in animals exposed to the mutant (B′). Significantly different values are indicated as: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

FIGURE 6

The effect of light organ colonization on CBF. Animals were inoculated with 5,000 CFU Vibrio fischeri per ml of NSW for symbiotic treatment, or only natural seawater (NSW) for aposymbiotic treatment. For treatments longer than 0.5 h, animals were rinsed twice in NSW after 3 h of exposure to the inoculum of V. fischeri and maintained in NSW for the remainder of experiment. CBF measurements were recorded at 0.5 h, 24 h, and 48 h. A one-way ANOVA and Tukey’s post hoc test were applied to the results (F_{5, 72} = 14.45, p < 0.0001). Animals (n) per treatment: 0.5 h aposymbiotic, n = 12; 24 h aposymbiotic, n = 10; 48 h aposymbiotic, n = 11; 0.5 h symbiotic, n = 19; 24 h symbiotic, n = 12; 48 h symbiotic, n = 13. Significantly different values are indicated as such: *p < 0.05; **p < 0.01; ns, not significant.