Development and Symbiosis Establishment in the Cnidarian Endosymbiosis Model Aiptasia sp

Abstract

Symbiosis between photosynthetic algae and heterotrophic organisms is widespread. One prominent example of high ecological relevance is the endosymbiosis between dinoflagellate algae of the genus Symbiodinium and reef-building corals, which typically acquire symbionts anew each generation during larval stages. The tropical sea anemone Aiptasia sp. is a laboratory model system for this endosymbiosis and, similar to corals, produces non-symbiotic larvae that establish symbiosis by phagocytosing Symbiodinium from the environment into the endoderm. Here we generate the first overview of Aiptasia embryogenesis and larval development and establish in situ hybridization to analyze expression patterns of key early developmental regulators. Next, we quantify morphological changes in developing larvae and find a substantial enlargement of the gastric cavity over time. Symbiont acquisition starts soon after mouth formation and symbionts occupy a major portion of the host cell in which they reside. During the first 14 days of development, infection efficiency remains constant while in contrast, localization of phagocytosed symbionts changes, indicating that the occurrence of functional phagocytosing cells may be developmentally regulated. Taken together, here we provide the essential framework to further develop Aiptasia as a model system for the analysis of symbiosis establishment in cnidarian larvae at the molecular level.

Figure 1. Development of Aiptasia.

(a) Overview of Aiptasia embryonic and larval development using differential interference contrast (DIC) microscopy. *indicates the blastopore; hpf = hours post fertilization; dpf = days post fertilization. (b) Representative confocal microscopy images of Aiptasia blastula (A–A”) and gastrula (B–B”). The left panels (A and B) show merged images of Hoechst-stained nuclei (blue) and phalloidin-stained F-actin (green), the middle panels (A’ and B’) only actin, and the right panels (A” and B”) only nuclei. (c) Gene expression patterns of key classical developmental regulators in Aiptasia larvae ∼24 hpf using in situ hybridization.

Figure 2. Morphological changes of the larval endoderm during development.

(a) Representative confocal microscopy images of Aiptasia larvae 48 hpf (A–A”), 8 dpf (B–B”), and 12 dpf (C–C”). The upper row (A–C) shows merged images of Hoechst-stained nuclei (blue) and phalloidin-stained F-actin (green), the middle row (A’–C’) only actin, and the lower row (A”–C”) only nuclei. The endoderm (en), ectoderm (ec), gastric cavity (gc), pharyngeal ectoderm (phec), and mesoglea (m) are indicated. (b) Schematic of larva with colored lines indicating positions of measurement of morphological features in (c–e). n = 23 for larvae 48 hpf and n = 22 for larvae 10 dpf. (c) Quantification of change in gastric cavity area between larvae 48 hpf and 10 dpf. Error bars are SEM, ***p < 0.001 as determined by Student’s t-test for unpaired data. (d) Quantification of change in larval length and width between larvae 48 hpf and 10 dpf. Error bars are SEM. (e) Quantification of change in thickness of the ectoderm (ec), endoderm (en1, en2), and pharyngeal width (ph) between larvae 48 hpf and 10 dpf. Error bars are SEM, ***p < 0.001 as determined by Student’s t-test for unpaired data. (f) Representative confocal microscopy images of Aiptasia larvae 48 hpf and 10 dpf showing phalloidin-stained F-actin to mark the cell outlines. Each image comprises z-projections of multiple planes of the endoderm. Below are corresponding higher-magnification images of endodermal cells. (g) Quantification of endodermal cell sizes of larvae 48 hpf and 10 dpf from images as shown in (f). Error bars are SEM, ***p < 0.001 as determined by Student’s t-test for unpaired data, n = 56 cells for larvae 48 hpf (5 larvae) and n = 82 cells for larvae 10 dpf (5 larvae). (h) Schematic of larvae summarizing morphological changes: younger larvae (48 hpf) have a small gastric cavity, a thick endoderm with columnar cells, and a pronounced pharyngeal ectoderm when compared to older larvae (10 dpf), which have a bigger gastric cavity, flattened endodermal cells, smaller pharyngeal ectoderm, and more pronounced mesoglea.

Figure 3. Symbiosis establishment during larval development.

(a) Quantification of symbiont uptake efficiencies in Aiptasia embryos and early planula larvae: early (<32-cell-stage) embryos were exposed to a constant environmental supply of Symbiodinium strain SSB01 (10,000 algae/ml) and subsets were sampled at the times indicated (hpf) to assess infection efficiency. Representative DIC images are shown below each timepoint. Error bars are SEM, n = 3 replicate experiments. (b) Quantification of symbiont uptake efficiencies for larvae between 2 and 20 dpf: larvae at the ages indicated (dpf) were incubated with Symbiodinium strain SSB01 (10,000 algae/ml) for four days, after which infection efficiency was assessed. Error bars are SEM, n = 3 replicate experiments. (c) Quantification of symbiont uptake efficiencies after four days exposure for larvae 6–7 dpf at increasing algal concentrations. Error bars are SEM, n = 3 replicate experiments. (d) Quantification of comparison of uptake efficiency between SSB01 algae and inert fluorescent beads for larvae 4 dpf after four days exposure. Error bars are SEM, ***p < 0.001 as determined by Student’s t-test for unpaired data, n = 3 replicate experiments. (e) Representative fluorescence microscopy images of phagocytosed symbionts in endodermal cells of larvae 8 dpf. Hoechst-stained nuclei are shown in blue, phalloidin-stained F-actin to mark cell outlines in green, and endogenous autofluorescence of algal chlorophyll in red. Note that algae exhibit strong autofluorescence in all channels. (f) Representative confocal microscopy images of larvae 10 dpf with or without symbionts. Fluorescence channels are as in (e).

Figure 4. Change in localization of symbiosis establishment during development.

(a–c) After 24 h exposure to symbionts (100,000 algal cells/ml), larvae 48 hpf and 10 dpf were scored for infection efficiency and average number of algal cells per larva. n = 3 replicate experiments (a). Additionally, the localization of the algal cells in the aboral or oral endoderm in each larva was recorded: representative DIC microscopy images are shown in (b) and quantification in (c). n = 3 replicate experiments, ≥30 larvae per experiment. Error bars are SEM, *p < 0.05 as determined by Student’s t-test for unpaired data.