Evolutionary origins of sensation in metazoans: functional evidence for a new sensory organ in sponges

Abstract

Background: One of the hallmarks of multicellular organisms is the ability of their cells to trigger responses to the environment in a coordinated manner. In recent years primary cilia have been shown to be present as 'antennae' on almost all animal cells, and are involved in cell-to-cell signaling in development and tissue homeostasis; how this sophisticated sensory system arose has been little-studied and its evolution is key to understanding how sensation arose in the Animal Kingdom. Sponges (Porifera), one of the earliest evolving phyla, lack conventional muscles and nerves and yet sense and respond to changes in their fluid environment. Here we demonstrate the presence of non-motile cilia in sponges and studied their role as flow sensors.

Results: Demosponges excrete wastes from their body with a stereotypic series of whole-body contractions using a structure called the osculum to regulate the water-flow through the body. In this study we show that short cilia line the inner epithelium of the sponge osculum. Ultrastructure of the cilia shows an absence of a central pair of microtubules and high speed imaging shows they are non-motile, suggesting they are not involved in generating flow. In other animals non-motile, 'primary', cilia are involved in sensation. Here we show that molecules known to block cationic ion channels in primary cilia and which inhibit sensory function in other organisms reduce or eliminate sponge contractions. Removal of the cilia using chloral hydrate, or removal of the whole osculum, also stops the contractions; in all instances the effect is reversible, suggesting that the cilia are involved in sensation. An analysis of sponge transcriptomes shows the presence of several transient receptor potential (TRP) channels including PKD channels known to be involved in sensing changes in flow in other animals. Together these data suggest that cilia in sponge oscula are involved in flow sensation and coordination of simple behaviour.

Conclusions: This is the first evidence of arrays of non-motile cilia in sponge oscula. Our findings provide support for the hypothesis that the cilia are sensory, and if true, the osculum may be considered a sensory organ that is used to coordinate whole animal responses in sponges. Arrays of primary cilia like these could represent the first step in the evolution of sensory and coordination systems in metazoans.

Figure 1

Cilia are found on the epithelia lining the osculum. a. The sponge Ephydatia muelleri in the lake, and grown in the lab viewed from the side (upper inset) and from above (lower inset). The oscula (white arrows) extend upwards from the body. b, c, Scanning electron micrographs show cilia arise from the middle of each cell along the entire length of the inside of the osculum; b the lining of the osculum with cilia on each cell (inset shows an osculum removed from the sponge and sliced in half longitudinally); c, two cilia arise from each cell. d, e, Cilia in the oscula labeled with antibodies to acetylated α-tubulin (green), nuclei with Hoechst (blue, n), actin with phalloidin (red). f. A 3D surface rendering illustrates how the cilia arise just above the nucleus of the cell. Scale bars a 5 mm; inset 1 mm; b 20 μm; inset 100 μm c, 1 μm d, 20 μm e, f 5 μm.

Figure 2

Cilia are non-motile and are oriented perpendicular to the direction of water flow in the osculum. a. Serial longitudinal sections (86 nm apart) show each cilium arises just above the cell nucleus (n) from simple basal bodies (bb); no links between the bases of the ciliary pair were found. b. In cross-section the cilium lacks a central microtubule pair in contrast to the cross section of a flagellum from a choanocyte chamber. c. Cilia pairs are aligned parallel to the long axis of the cells in the osculum, and both the cilia pairs and the cells’ long axes lie perpendicular to the direction of water flow (shown by the blue arrow) at 345.12 ± 4.72° (mean ± SE) (rose diagram: H_A:0°; V = 0.841; p < 0.001; n = 49). d. Still images from high-frequency time-lapse imaging of live cilia (arrows) labeled with FM1-43 (see Additional file 2: Movie S1). Scale bars: a, 500 nm b, 100 nm c, 10 μm d, 20 μm.

Figure 3

Cationic channel blockers reduce the ‘sneeze’ response. a. The sponge ‘sneeze’ behaviour involves contraction of the osculum (white arrows), inflation, then contraction of canals (black arrows) and recovery (bar shows canal diameter). b. Neomycin sulfate (red) and FM1-43 (blue) reduce the peak amplitude of the behaviour in E. muelleri (n = 8; p < 0.001). Gd³⁺ (solid green) eliminated all response (n = 3; p = 0.015), but after recovery for 24 h the sponge response was even greater than before (dotted green). c, d All three compounds caused lengthening of cilia relative to controls (left), but had no effect on choanocyte flagella (bottom right) in E. muelleri (*significance at p < <0.001; error bars show ± SE). Scale bars: a, 1,000 μm c, 10 μm.

Figure 4

Cilia are specifically involved in the sponge behaviour. a. In contrast to Neomycin sulfate (solid red) which eliminates the ‘sneeze’ response (n = 3, p = 0.035), the calcium channel blocker Verapamil (dotted red) does not affect amplitude of the sneeze behaviour in S. lacustris (n = 5, p = 0.573). b. Texas-Red Neomycin sulfate conjugate (red) and YO-PRO1 (green) selectively label cells in the osculum. c. A 20 hr treatment in chloral hydrate eliminates the sneeze behaviour in S. lacustris (solid green; n = 5, p = 0.004), which does not return until more than 3 days after recovery (dotted green; n = 5, 24 hr washout p = 0.003, 72 hr washout p = 0.018, 120 hr washout p = 0.864)). d-f(SEM) d’-f’(fluorescence). Cilia are removed by chloral hydrate treatment; S. lacustris 0 hr (d, d’), 20 hr (e, e’), and 70 hr (f, f’) treatment in chloral hydrate. g. The sneeze behaviour in S. lacustris cannot be triggered when the osculum is removed (solid blue; n = 3, p = 0.010) until it has fully regrown (dotted blue; n = 3, p = 0.275). h. Ciliated cells on the surface of E. muelleri 8 hr post osculum removal and (i) in the newly formed osculum 24 hr post osculum removal. Ciliated cells do not become labeled with EdU until after the osculum has regrown suggesting they arise by migration of newly formed mesohyl cells which differentiate into ciliated pinacocytes. Cilia are labeled with acetylated α-tubulin (red), nuclei with Hoechst (blue), and newly synthesized DNA with EdU (green). Scale bars: b, 50 μm inset 10 μm d, e, 5 μm d’, e’, f, f’, h, i 10 μm.

Figure 5

Phylogenetic analysis of sponge TRP genes. a. Evolutionary relationships of sponge TRP Type I and II genes, values in the nodes indicate Boostrap Support and Posterior Probabilities (see Methods); sponge sequences are in bold. b. Domain diagrams showing the PKD channel domain, transmembrane domain (TM), EF hand domain, and ion transport domains for the pkd2 genes from mouse, Mus musculus; Cca, Corticium candelabrum (Homoscleromorpha); Cel, Caenorhabditis elegans; Sla, Spongilla lacustris (Demospongiae); Sco; Sycon coactum (Calcarea); Ava, Aphrocallistes vastus (Hexactinellida); Cre, Chlamydomonas reinhardtii, and 3D models of the proteins from mouse, Corticium, Sycon, and Chlamydomonas. c. Alignment of bilaterian, cnidarian and sponge TRP sequences showing the TRP domain and TRPbox (Hsap, Homo sapiens; Mmus, Mus musculus; Spur, Strongylocentrotus purpuratus; Cint, Ciona intestinalis, Sko, Saccoglossus kowaleskii, Lforb, Loligo forbesi, Bflo, Branchiostoma floridae, Sman, Schistosoma mansoni, Nvec, Nematostella vectensis). For the full tree and alignment see Additional file 1: Figures S2-S4.