Morphological characterization of virus-like particles in coral reef sponges

Abstract

Marine sponges host complex microbial consortia that vary in their abundance, diversity and stability amongst host species. While our understanding of sponge-microbe interactions has dramatically increased over the past decade, little is known about how sponges and their microbial symbionts interact with viruses, the most abundant entities in the ocean. In this study, we employed three transmission electron microscopy (TEM) preparation methods to provide the first comprehensive morphological assessment of sponge-associated viruses. The combined approaches revealed 50 different morphologies of viral-like particles (VLPs) represented across the different sponge species. VLPs were visualized within sponge cells, within the sponge extracellular mesohyl matrix, on the sponge ectoderm and within sponge-associated microbes. Non-enveloped, non-tailed icosahedral VLPs were the most commonly observed morphotypes, although tailed bacteriophage, brick-shaped, geminate and filamentous VLPs were also detected. Visualization of sponge-associated viruses using TEM has confirmed that sponges harbor not only diverse communities of microorganisms but also diverse communities of viruses.

Keywords: GBR; Great Barrier Reef; Marine sponges; Red Sea; TEM; Transmission Electron Microscopy; VLP; Virus.

Figure 1. Representative morphotypes of virus-like particles associated with GBR and Red Sea sponges.

GBR sponge species: (A, J, K, L) C. foliascens, (B) Xestospongia sp., (C, F, G) E. isaaci, (E) C. schulzei. Red Sea sponge species: (D, H) S. carteri, (K) Amphimedon ochracea. TEM preparation method: (A, J, K) ultrathin sections of sponge tissue, (B–I, L) viral purification via filtration of sponge mucus. Scale bar: 200 nm. Black arrows indicate the viral tail and white arrows indicate the VLPs.

Figure 2. Representative morphotypes of virus-like particles associated with GBR sponges.

Sponge species: (A) C. foliascens, (B, C) Stylissa carteri, (D) Xestospongia sp., (E–H) Pipestela candelabra, (I–K) Lamellodysidea herbacea, (L) C. schulzei. TEM preparation method: (A, H–K) viral purification via filtration of sponge mucus, (B–F, L) viral purification via CsCl gradient centrifugation, (G) ultrathin sections of sponge tissue. Scale bar: 200 nm.

Figure 3. Representative morphotypes of virus-like particles associated with GBR and Red Sea sponges.

GBR sponge species: (A, B) C. schulzei, (C) Cymbastella marshae. Red Sea sponge species: (D, E) C. foliascens, (F–H) S. carteri, (I) Xestospongia testudinaria, (J–L) Hyrtios erectus. TEM preparation method: (A, D–E, I–L) ultrathin sections of sponge tissue, (B, C, F–H) viral purification via filtration of sponge mucus. Scale bar: (A–C, E–L) 200 nm, (D) 500 nm. Black arrows indicate the VLPs.

Figure 4. Representative morphotypes of virus-like particles associated with GBR and Red Sea sponges.

GBR sponge species: (A) Mycale sp., (B) C. foliascens, (C, D) Xestospongia sp., (E) C. schulzei. Red Sea sponge species: (F, G) C. foliascens, (H, I) S. carteri, (J–L) Xestospongia testudinaria. TEM preparation method: (A, B, D, E, H, I) viral purification via filtration of sponge mucus, (C) viral purification via CsCl gradient centrifugation, (F, G, J–L) ultrathin sections of sponge tissue. Scale bar: (A–E, H, I) 200 nm, (F, G, J–L) 500 nm.

Figure 5. Representative morphotypes of virus-like particles associated with GBR and Red Sea sponges.

GBR sponge species: (C) Lamellodysidea herbacea, (I) C. foliascens. Red Sea sponge species: (A, B, H) Crella cyathophora, (D–G) Amphimedon ochracea, (J–L) Hyrtios erectus. TEM preparation method: (A, C, D) viral purification via filtration of sponge mucus, (B, E–L) ultrathin sections of sponge tissue. Scale bar: (D) 100 nm, (A–C, E, H–L) 200 nm, (F, G) 5 µm. ECM: External Cell Matrix, om: outer membrane, im: inner membrane, cm: core membrane, lb: lateral bodies; c: core, e: external membrane; b: bacterium. Black arrows indicate the VLPs.