Defending against pathogens – immunological priming and its molecular basis in a sea anemone, cnidarian

Cnidarians, in general, are long-lived organisms and hence may repeatedly encounter common pathogens during their lifespans. It remains unknown whether these early diverging animals possess some type of immunological reaction that strengthens the defense response upon repeated infections, such as that described in more evolutionary derived organisms. Here we show results that sea anemones that had previously encountered a pathogen under sub-lethal conditions had a higher survivorship during a subsequently lethal challenge than naïve anemones that encountered the pathogen for the first time. Anemones subjected to the lethal challenge two and four weeks after the sub-lethal exposure presented seven- and five-fold increases in survival, respectively, compared to the naïve anemones. However, anemones challenged six weeks after the sub-lethal exposure showed no increase in survivorship. We argue that this short-lasting priming of the defense response could be ecologically relevant if pathogen encounters are restricted to short seasons characterized by high stress. Furthermore, we discovered significant changes in proteomic profiles between naïve sea anemones and those primed after pathogen exposure suggesting a clear molecular signature associated with immunological priming in cnidarians. Our findings reveal that immunological priming may have evolved much earlier in the tree of life than previously thought.


Supplemental
: Percent survival of Exaiptasia pallida to the exposure of the bacterial pathogen Vibrio coralliilyticus over ten days. A) Percent survival of E. pallida anemones under three different concentrations of V. coralliilyticus inoculum: 10 6 , 10 7 , and 10 8 CFU ml -1 at 30˚C. The inoculum concentration of 10 8 CFU ml -1 was chosen for the priming experiment since it showed the most consistent mortality results than using other inoculum concentration. B) Percent survival of E. pallida to the exposure of Vibrio coralliilyticus inoculum at different temperatures over ten days. Challenges were conducted at a concentration inoculum of 10 8 CFU ml -1 at both 25 and 30˚C. One hundred percent mortality was recorded at the bacterial challenge conducted at 30˚C by day seven of the experiment. Importantly, an increased temperature alone with no bacterial challenge did not cause mortality. Additionally, a bacterial challenge at 25˚C did not result in mortality over the ten-day experiment. These results indicated that bacterial infections of Vibrio coralliilyticus require to be performed at 30˚C, and that this experimental temperature has no negative effect on survivorship of Exaiptasia pallida anemones.
Supplemental Figure 2: Percent survival of Exaiptasia pallida anemones varying the exposure time to V. coralliilyticus at a dose of 10 8 CFU ml -1 : ten days, three days, and control. Results indicate that sea anemones show the same degree of survival to controls when only exposed to the bacterial pathogen, V. coralliilyticus for three days. The anemones that were exposed to the pathogen for ten days started dying after the fourth day of exposure. These results suggested that a 3-day exposure to the pathogen at 1x10 8 CFU ml -1 is considered a sub-lethal treatment and was used as the priming condition in the immunological experiments in this study. Exposure for more than four days is considered lethal.
Supplemental Figure 3: Relative Abundance of Vibrio coralliilyticus load in infected Exaiptasia pallida anemones estimated through specific quantitative PCR amplification of the pathogen 16S rDNA. A) Relative abundance of V. coralliilyticus load in infected anemones during the first four days after the completion of the sub-lethal exposure. The relative abundance of the pathogen is defined as the amount of bacterial load on the anemone in comparison to a bacterial concentration of 1x10 8 CFU ml -1 . Day 0 represents anemones sampled immediately after being transferred to a new well for recovery at the completion of the sub-lethal exposure, Day 1 and Day 4 represent anemones sampled after one and four days following the completion of the sub-lethal exposure, respectively.
Results indicate that V. coralliilyticus is cleared by E.pallida anemones by day four following the completion of the sub-lethal exposure (ANOVA, p=0.04, Tukey HSD, p<0.05). Error bars indicate standard deviation. B) Relative abundance of V. coralliilyticus load anemones during the ten-day lethal exposure. Day 1, 3, 7, and 10 represent days when anemones were sampled during the ten-day lethal exposure. Error bars indicate standard deviation. Results indicate that a considerable amount of V. coralliilyticus load is present in the tissue of E. pallida throughout the ten-day lethal exposure. The highest pathogen load was detected during the first three days of the challenge and then it declined at day 7 suggesting some level of clearance by the few anemones that had survived at this time (ANOVA, p=0.01; Tukey HSD, p<0.05).
Supplemental Figure 4: CyDye switch, two dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of proteomes from naïve (N=3) and pathogen-primed (N=3) Exaiptasia pallida sea anemones. Naïve anemone samples were labeled with Cy3 (green) and primed anemone samples with Cy5 (red). Samples were then mixed and separated on analytical 2-D DIGE. The resulting gel was scanned and the merged image is shown where red proteins represent proteins whose expression is higher in the primed anemone tissue and green proteins represent proteins whose expression is higher in the naïve anemone tissue. The depicted gel is one example of the three replicated gels produced in this proteomic analysis. Circled and numbered spots represent proteins, which were most differentially expressed of which only those indicated by yellow circles were able to be analyzed using Mass-Spec and reported in Table 1