Endosymbiotic calcifying bacteria: a new cue to the origin of calcification in metazoa?

Abstract

Sponges show the highest diversity of associated bacteria among marine invertebrates. Immunological evidence traces the origin of the sponge bacterial symbioses to the Precambrian era. Hence, sponges appear to be ideally suited for studying the evolutionary origins of prokaryote-metazoan associations. Sponges produce either calcareous or siliceous skeletons, which only coexist in a relict group of demosponges, the sclerosponges. We report here, for the first time, intensive calcification in nonsclerosponge siliceous demosponges. Calcification is mediated by endosymbiotic bacteria (calcibacteria) located in archeocyte-like sponge cells. These calcibacteria are devoid of bacterial walls and divide within sponge cells until they became surrounded by a calcitic sheet, being subsequently extruded to the sponge subectosomal (subepithelial) zone. Thousands of bacteria-produced calcitic spherules cover the surface of the host sponges, forming a cortex-like structure that mimics a rudimentary peripheral skeleton. Calcibacteria are vertically transferred to the sponge larvae during embryogenesis. Calcium detoxification may have generated this symbiotic association, with some additional benefits for the sponges, such as skeletal formation and deterrence from predation. This unique symbiosis holds implications for sponge biology and may advance discussions on the role of bacteria in early biocalcification processes in metazoans.

Figure 1

Sponge species harboring calcibacteria. (A) Hemimycale columella (Atlanto-Mediterranean). (B) Hemimycale sp. (Indo-Pacific). (C) Hemimycale arabica (Red Sea). (D) Whitish tinge of the sponge surface (arrows) due to calcibacteria accumulation; scale bar 2 mm. (E) Calcibacteriocytes (arrows) surrounding an embryo; (e) scale bar 50 μm. (F) Calcibacteria accumulation (white spots) in a 2-week-old recruit (rhagon); scale bar 1 mm.

Figure 2

Light microscopy pictures of calcibacteriocytes and calcibacteria. (A) Calcibacteriocytes full of refringent calcibacteria; scale bar 5 μm. (B) Released calcibacteria after crushing a living individual of Hemimycale columella; scale bar 10 μm. (C, D) CARD-FISH epifluorescence micrographs of H. columella sections, either labeled with bacteria-specific probe EUB338 (green) (C) or with archaea-specific probe ARCH915 (D); scale bar 40 μm. (E, F) Higher magnification of sponge section showing a calcibacteria-full calcibacteriocyte (arrows) either stained with DAPI (E)—see also sponge cell nuclei (n)—or labeled with bacteria-specific probe; (F) scale bar 5 μm.

Figure 3

TEM micrographs. (A) Early calcibacteria (arrows) within calcibacteriocyte vacuoles; scale bar 1μm. (B) Calcibacteria (cb) starting biocalcification (arrow points to calcification vesicles); scale bar 0.2 μm. (C) Calcibacteria division within a vacuole (arrows); scale bar 1 μm. (D) Calcibacteriocyte (cbc) with dividing calcified bacteria; scale bar 1 μm. The bacteria calcareous coat is not stained by the osmium tetroxide. (E) Calcified calcibacteriocytes, released to the sponge mesohyle; scale bar 1 μm. (F) Sponge larva with dividing calcibacteria, transmitted from a maternal calcibacteriocyte (cbc): larval cilia (c and arrows); larval pseudoepithelial cell; (e) scale bar 2 μm.

Figure 4

SEM micrographs and EDX of calcibacteria. (A) Energy dispersive X-ray Analysis (EDX) of the calcareous coat. (B) Dividing calcibacteria entrapped within the calcareous coat (SEM); scale bar 600 nm. (C) Broken calcibacteria showing the thin calcareous coat and the inner organic matter (SEM); scale bar 500 nm. (D) High density of calcibacteria released from broken calcibacteriocytes after squeezing the sponge (SEM); scale bar 1μm.