Multiple bacterial partners in symbiosis with the nudibranch mollusk Rostanga alisae

Abstract

The discovery of symbiotic associations extends our understanding of the biological diversity in the aquatic environment and their impact on the host's ecology. Of particular interest are nudibranchs that unprotected by a shell and feed mainly on sponges. The symbiotic association of the nudibranch Rostanga alisae with bacteria was supported by ample evidence, including an analysis of cloned bacterial 16S rRNA genes and a fluorescent in situ hybridization analysis, and microscopic observations. A total of 74 clones belonging to the phyla α-, β-, γ-Proteobacteria, Actinobacteria, and Cyanobacteria were identified. FISH confirmed that bacteriocytes were packed with Bradyrhizobium, Maritalea, Labrenzia, Bulkholderia, Achromobacter, and Stenotrophomonas mainly in the foot and notum epidermis, and also an abundance of Synechococcus cyanobacteria in the intestinal epithelium. An ultrastructural analysis showed several bacterial morphotypes of bacteria in epidermal cells, intestine epithelium, and in mucus layer covering the mollusk body. The high proportion of typical bacterial fatty acids in R. alisae indicated that symbiotic bacteria make a substantial contribution to its nutrition. Thus, the nudibranch harbors a high diversity of specific endo- and extracellular bacteria, which previously unknown as symbionts of marine invertebrates that provide the mollusk with essential nutrients. They can provide chemical defense against predators.

Figure 1

Morphology and localization of symbionts in Rostanga alisae. (A) Common view of the nudibranch in the natural habitat on the sponge. (B) Histological organization, longitudinal section. (C–E) are enlarged views of the areas indicated by the corresponding arrows: (C) Bacteria (b) in the mucus layer covering the body. Bacterial clusters (bc) in the epidermis of the notum (D) and foot (E). Nuclei (nu) of epithelial cells are visible. Combination of fluorescence microscopy and Phase microscopy, DNA stained with DAPI (blue fluorescence).

Figure 2

Maximum-likelihood tree of the 16S rRNA sequences of Rostanga alisae symbionts. The tree is based on the TIM3 + F + I + G4 model of nucleotide substitution. The numbers at the nodes are bootstrap percent probability values based on 10,000 replications (values below 75% are omitted). The number following the name of specimens (RF, RN, and RI) denotes the clone number. RF foot, RN notum, RI intestine.

Figure 3

Fluorescent in situ hybridization microscopy of bacteria forming bacteriocytes (bc) in the epidermis and cyanobacteria in the intestinal epithelium of Rostanga alisae. (A) Accumulation of bacteriocytes (bc) in the skin fold between the foot and notum, transverse section of the body. Red fluorescence of bacteria hybridized with Cy3 universal UEB388 probe set. Green fluorescence of bacteria hybridized with bacteria-specific probes for (B) Achromobacter, (C) Stenotrophomonas, (D) Labrenzia, (E) Maritalea, (F) Bradyrhizobium, (G) Burkholderia, (H,I) Synechococcus (arrows). DNA stained with DAPI (blue fluorescence).

Figure 4

Endosymbiotic bacteria housed in bacteriocytes of Rostanga alisae. (A) Cells of integument epithelium of the foot containing bacteriocytes with several bacterial morphotypes of bacteria. Phagosome (ph) is visible. (B) Bacteriocyte containing curved-rod bacteria with lophotrichous flagella (fl). Vacuoles with electron-transparent contents are visible in the cytoplasm of bacteria. (C) Bacteriocytes containing rod-shaped bacteria with flagella (black asterisk) and without flagella (white asterisk). (D) Dividing flagellate bacterium in the bacteriocyte (arrow). (E) Bacteriocyte containing large rod-shaped bacteria, which forming a chain as a result of incomplete division. Arrows show the contacts of bacteria. Nucleus (nu) of the epithelial cell is visible. (F) Higher magnification of bacteria of this morphotypes indicated in (E). A tortuous outer membrane, extensive nucleoid zone, and vacuoles are visible. Arrows show the contact points of the outer membranes of adjacent individuals.

Figure 5

Endosymbiotic single bacteria of Rostanga alisae, TEM images. (A) Numerous Single gram-negative bacteria within secondary vacuole (light arrows) in the apical zone of the cytoplasm of the epithelium of the foot. As a result of division (dark arrow indicates a dividing bacterium), bacteriocytes contain two or more bacteria. Cilia (c), phagosome (ph), mitochondria (m), and nucleus (nu) of epithelial cells are visible. (B,C) Cyanobacteria (arrows) in the epithelium of the notum and (D) foot; a developed polysaccharide capsule and concentrically arranged membranes of the thylakoid type (th) are visible. (E) Different morphotypes of cyanobacteria containing inclusions in the intestinal epithelium cells: bacteria with a developed capsule (light arrow); bacteria with a narrow capsule (dark arrow). The apical surface of epithelial cells has microvilli (mv).

Figure 6

TEM images of epithelium cells and adjacent mucous layer of Rostanga alisae inhabited by exosymbiontic bacteria. (A) Bacteria having a long contact stalk-like extension directed towards the apical membrane of epithelial cells (white arrow). Dark arrows indicate similar bacteria, which are present both in the mucous layer and in vacuoles near the apical membrane of the integumentary epithelial cells. (B) The bacterium with a long process (dark arrows) in contact with the surface of the microvilli (cross section) of the integumentary epithelial cells. (C) Long rods localized between the microvilli (mv) of the integumentary epithelial cells. The arrow indicates the site of adhesion of the bacterial cell to the outer cell membrane. (D) Large bacteria with numerous electron-translucent vacuoles (dark arrow) (E) Processes of branching bacteria on the surface and in the cytoplasm of cilliated cells of the foot epidermis (dark arrows); branched processes are visible bearing spore-like vesicles on the surface (light arrow). Cilia (c) and microvilli (mv) are visible.

Figure 7

Distribution of the fatty acids among the notum and intestine of Rostanga alisae and its prey sponge Ophlithaspongia pennata (mean ± SD; n = 7). (A) Composition of principal fatty acids. (B) Composition of odd and branched fatty acids (OBFA) as markers of bacteria. Data on fatty acid composition and significant differences (one-way ANOVA) in their concentration between the nudibranch and its prey sponge are provided in Supplementary Table S3.