General observations of plastid dynamics within the digestive diverticula of <i>E. chlorotica</i> developing juveniles (<9 dpm).

<p>Many cellular observations in juveniles were in agreement with historical work on adult <i>E. chlorotica</i>, with the notable exception of massive LD accumulation in juvenile animals. (A) Numerous plastids accumulated within the cells of the digestive diverticula both surrounded by a membrane (arrow) or in apparent direct contact with the host cytosol (arrowhead). (B) Accumulation of the plastids corresponded with lipids, while the lumen of the animals remained relatively clear of debris. (C) A rare degraded plastid (arrow) observed within the lumen. (D) One rare example of potential phagocytosis of an algal plastid by the digestive cells. LD: lipid droplet, P: plastid, Lu: lumen, N: <i>E. chlorotica</i> nucleus.</p

Representative images of the digestive diverticula of juvenile <i>E. chlorotica</i> allowed to feed on <i>V. litorea</i> for 5 days prior to being starved of food ( = transient kleptoplasty).

<p>Animals were starved for 1 day (A), 3 days (B) or 7 days (C). The same age control animals, allowed to feed continuously, are in the corresponding position in the bottom panels (D–F). Plastid decay and LD degradation are apparent in the animals removed from food in comparison to the same age controls. LD: Lipid droplet, P: plastid, Lu: lumen, N: <i>E. chlorotica</i> nucleus, Ps: phagosome; arrows indicate decaying plastids, black marks in F are staining artifacts.</p

Lipid droplets in the digestive diverticula of <i>E. chlorotica</i> as observed using light (A,B) confocal (C) and transmission electron (D) microscopy.

<p>Green fluorescence in panel C is from the neutral lipid fluorescent label BODIPY 505/515 (Molecular Probes) verifying these abundant refractive bodies as lipids, and the red is chlorophyll autofluorescence. Animals in these image range in age from 4 dpm (A,B) to >6 months (C,D). Arrows (A) and LD: lipid droplet, P: plastid, Lu: lumen, N: <i>E. chlorotica</i> nucleus.</p

Anatomy of the sacoglossan mollusc <i>Elysia chlorotica</i>.

<p>(A) Sea slug consuming its obligate algal food <i>Vaucheria litorea</i>. Small, punctate green circles are the plastids located within the extensive digestive diverticula of the animal. (B) A defined tubule of the digestive diverticula extending into the parapodial region of the animal (arrow). The digestive system consists of densely packed tubules that branch throughout the animal's body. Each tubule is made up of a layer of single cells containing animal organelles and numerous algal plastids. This cell layer surrounds the lumen. (C) Magnified image of the epidermis of <i>E. chlorotica</i> showing densely packed plastids. The animals are light grey in color without their resident plastids, which contribute chlorophyll to render the sea slugs bright green.</p

Transmission electron micrographs of <i>E. chlorotica</i> illustrating the acquisition of both lipids and plastids upon feeding.

<p>(A,B) Aposymbiotic <i>E. chlorotica</i> at 1 dpm with neither plastids or lipids present in the digestive tissue. (C,D) Fed <i>E. chlorotica</i> at 1 dpm with large lipid accumulation and visible intracellular plastids. LD: lipid droplet, P: plastid, Lu: lumen, N: <i>E. chlorotica</i> nucleus, Ep: epidermis, R: radula.</p

Adult <i>E. chlorotica</i> (reared in the laboratory for 11 months and fed <i>V. litorea</i>) were used to compare the effects of food removal on the digestive diverticula of mature individuals.

<p>Starvation of adult animals showed no obvious reduction in lipid or plastid content. (A) Adult animal starved for 2 wk. (B) Adult animal fed continuously. LD: lipid droplet, P: plastid, Lu: lumen, N: <i>E. chlorotica</i> nucleus.</p

Representative pigment composition of animals that were raised in A) 24L, B) 12L∶12D, or C) 24D conditions and provided <i>V. litorea</i> for 4 wk before starvation for 4,6, and 8 wk.

<p>Animals were scored based on a color rubric ranging from dark green to grey, with movement away from dark green indicative of loss of plastids. There was a greater percentage of animals observed with pigment loss in the 24D (C) and 24L (A) conditions, than in 12L∶12D (B). Dotted line indicates when food was removed from all treatments.</p

Representative TEM images of LD dynamics during the initial 2<i>E. chlorotica</i> suggesting the source of lipid accumulation in the animal is the algal plastids.

<p>All images are of fed controls. Accumulation of LD within the plastids is visible in every panel, but most clearly in A–C. Exudation of lipids from the plastids is suggested by panels B–G. Accumulation of large intracellular LD is illustrated in F and G, and intra-lumenal LD in H and I. LD: lipid droplet, P: plastid, Lu: lumen, N: <i>E. chlorotica</i> nucleus; arrows indicate decaying plastids.</p

Representative images of the digestive diverticula of juvenile <i>E. chlorotica</i> allowed to feed on <i>V. litorea</i> for 7 days prior to being starved of food ( = permanent kleptoplasty).

<p>Animals were starved for 1(A), 3 d (B) or 7 d (C). The same age control animals, allowed to feed continuously, are in the corresponding position in the bottom panels (D–F). Starved and fed animals show very similar patterns. LD: lipid droplet, P: plastid, Lu: lumen, N: <i>E. chlorotica</i> nucleus; arrows indicate decaying plastids.</p

S2, Functional enrichments.

Significant functional enrichment in gene sets that were otherwise informative in understanding inter-study differences. First four data columns represent a contingency table with "S" standing for "set (of genes)" and "C" standing for "Category (functional)". A Fisher's exact test was used to determine significance, and the false discovery rate calculated using the Benjamini Yekutili method. Remaining columns supply information on the functional category, details of gene set, and direction of enrichment