The Journal of Experimental Biology

The Journal of Experimental Biology

584

region of cilia (Fig. 2J) (Leys and Degnan, 2001; Maldonado et al.,

2003). The pigment inclusions are intracellular, and appear to lie in

a cell adjacent to the ciliated sensory cell (Fig. 2K). The simplest

explanation for the ‘steering’ of the larva is that each cell responds

independently to changes in light intensity as the larva rotates

through the water (Leys and Degnan, 2001). But some larvae have

cytoplasmic bridges between the protrusions containing the pigment

(e.g. Maldonado et al., 2003), so some sort of more rapid

communication between the pigment cells should not be ruled out

because cytoplasmic bridges usually occur in tissues that need to

maintain quicker communication (e.g. for coordinating

developmental processes in sperm or in the embryo).

The photo pigment in the Amphimedon queenslandica larva has

been studied more closely and is thought to be a cryptochrome with

sensitivity at around 450 nm (Leys et al., 2002). Two cryptochromes

AqCry1 and AqCry2 were purified from A. queenslandica and one,

AqCry2, showed sensitivity to blue light and was expressed in a

region around the pigment ring where the light sensitive cilia occur

at the posterior pole of the larva (Fig. 2L) (Rivera et al., 2012). The

interpretation is that the Cry genes encode proteins that are located

in the ciliated cells in the larva, but further work using antibodies is

needed to confirm this. It is possible that other proteins are involved

in the light response of the larva, because a 600 nm peak was

suggested to be due to an opsin-like molecule [see fig. 7 in Leys and

Meech (Leys and Meech, 2006)]. So far, no true opsin has been

found in either the Amphimedon queenslandica or  Oscarella

carmela genomes nor in any transcriptome from sponges (Feuda et

al., 2012).

Other sponge larvae also have phototactic behaviour (Maldonado

et al., 2003; Collin et al., 2010). Amphiblastula larvae of calcareous

sponges show negative phototaxis (Elliott et al., 2004) and have

curious ‘cross cells’ which express Smad1/5 (Leininger et al., 2014)

as well as SoxB (Fortunato et al., 2012), genes that are also

expressed in vertebrate sensory systems. In early work, Tuzet

suggested that the cross cells were involved in photosensation

(Tuzet, 1973), but no experiments have tested this. The absence of

any opsins in sponges is curious because opsins are known from

plants and fungi (microbial, type I opsins) and are thought to be

convergent with animal type II opsins (Heintzen, 2012). At least two

rhabdomeric (type II) opsins have been found in ctenophores

(Schnitzler et al., 2012). Were opsins, like nerves, also lost in

sponges?

Conducting pathways and effectors

If sensory cilia receive signals, how is the signal transmitted through

the sponge and what is the effector? In glass sponges the syncytial

REVIEW

The Journal of Experimental Biology (2015) doi:10.1242/jeb.110817

Fig. 2. Sensory cilia in sponges. (A) Transparent raised excurrent canals leading to the osculum (arrow) in Spongilla lacustris encrusting on a branch in a

lake. (B) The osculum (arrow) of a small lab-hatched individual of Spongilla lacustris. (C,D) Scanning electron micrographs of cilia (arrows) on the inner

epithelium of an osculum cut open lengthwise. (E,F) Immunofluorescence of the whole osculum (E), and a single endopinacocyte (F) showing cilia labelled with

the styrl dye FM 1-43 (green, arrows), nuclei (blue) and actin (red) (images, D. Ludeman). (G,H) Transmission electron micrograph of a section through the

osculum showing the base of one cilium arising just above the nucleus (nu); inset shows a cross section of the cilium with no clear central pair of microtubules.

(I) Scanning electron micrograph of the larva of Amphimedon queenslandica showing swimming cilia forming metachronal waves (arrows) and long posterior

cilia (right). (J) Response of the long posterior cilia in A. queenslandica to changes in light intensity: (I) bent when suddenly dark and (II) straightened when

suddenly light (from Leys et al., 2002). (K) Transmission electron micrograph through the pigment granules (pg) and long posterior cilia of the A. queenslandica

larva. (L) Expression of the AqCRY2 gene in two developmental stages (i,ii) at the posterior pole of the A. queenslandica larva (from Rivera et al., 2012). Scale

bars: 5 mm (A); 50 μm (B); 10 μm (C,E,F); 1 μm (D); 2 μm (G); 100 nm (G, inset); 500 nm (H); 100 μm (I,J); 5 μm (K).

---

The Journal of Experimental Biology

tissues transmit electrical signals, and the effectors are the flagella

of choanocytes, which stop beating. Cellular sponges have no

electrical signals, and are not known to arrest their flagella beating,

so the effectors are contractile cells that reduce the size of the canals

and chambers, effectively reducing flow into and through the

sponge. Earlier workers identified the effectors of contractions in

sponges as a type of smooth muscle cell called a myocyte (Bagby,

1966; Prosser, 1967); it was thought that these could be both in the

mesohyl and epithelium. Recent work has referred to them as

actinocytes and there is some evidence that actinocytes are largely

epithelial, i.e. are pinacocytes, and that mesohyl cells play a passive

role in contractions (Nickel et al., 2011). Where canals are wide,

‘sphincters’ made from one or more specialized pinacocytes arise

from the canal epithelium, allowing the sponge to constrict a portion

of the canal. In other places, sieve cells function in the same way to

reduce the dimensions of the incurrent space. In Tethya wilhelma,

for example, a sieve-like cell (sometimes two) forms the apopyle or

excurrent passage of chambers and this cell expresses genes for

myosin (Steinmetz et al., 2012).

Whereas pinacocytes are stationary and maintain contact with

neighbours via adherens and septate junctions, many cells in the

sponge mesohyl are in constant motion and do not seem to stay in

contact with epithelia or with other cells for long. Both Prosser

(Prosser, 1967) and Adams et al. (Adams et al., 2010) have shown

that sponges control the ionic milieu of the extracellular space, so

signalling is expected to be juxtacrine – being released from one cell

to trigger a response in a neighbouring cell without direct passage

of material from cell to cell. In fact, few examples exist of direct

exchange of materials between sponge cells and this seems to be one

of the main puzzles given the description of a near complete set of

scaffolding proteins involved in post-synaptic densities (PSDs) in

the Amphimedon queenslandica genome (Sakaraya et al., 2007; Alié

and Manuel, 2010) as well as in other sponge transcriptomes

(Riesgo et al., 2014).

Numerous ultrastructural studies on different sponges show

regions of density between neighbouring cells – cells apparently

exchanging large vesicles, some with distinct clathrin-coated pits

(Pavans de Ceccatty et al., 1970; Lethias et al., 1983) – but no

obvious synaptic structure with a post-synaptic density has been

found. Many PSD proteins are also found in unicellular eukaryotes

where there is clearly no pre-neuronal role (Burkhardt et al., 2014).

So a neuronal context is not necessarily implied by gene content.

But knowing whether PSD genes occur and function together in

sponges would help determine when components of a proper PSD

arose. In this vein, correlation analysis by Conaco et al. (Conaco et

al., 2012) suggested that although there is a lack of global co-

regulation of the entire set of PSD genes, small modules are co-

expressed. But there is some circularity in this reasoning, because

the same analysis suggests there is no co-regulation of epithelial

genes in sponges based on the fact that the authors did not consider

sponges to possess proper epithelia. A number of PSD genes

(Homer, CRIPT, DLG etc.) are expressed in globular cells of the

epithelium of the larva of Amphimedon queenslandica, which are

interpreted to be potential sensory cells receiving signal cues that

guide settlement behaviour (Sakaraya et al., 2007; Richards et al.,

2008). Normally PSDs are in the cell receiving the signal, not the

sensory cell, so their location in the globular cell of Amphimedon is

confusing. Globular cells in Amphimedon also express many other

genes [(e.g. NF-κB (Gauthier and Degnan, 2008); bHLH and Delta

(Richards et al., 2008); Frizzled (Adamska et al., 2010); TIRs

(Gauthier et al., 2010)] so experimental work is needed to determine

whether the gene expression is linked to sensory function.

Ionic physiology and signalling molecules

Only glass sponges (Hexactinellida) use electrical impulses to

rapidly send signals arresting the feeding current (Leys and Mackie,

1997). All attempts to determine the mechanism of contractions and

signal propagation in other sponges, including bath application of

chemicals, substitution of ions in the medium and triggering with

mechanical and electrical stimuli, so far show that electrical

signalling does not occur in cellular sponges. Loewenstein

(Loewenstein, 1967) reported that aggregating cells of Haliclona

spp. could pass current to one another in the presence of calcium and

magnesium, suggesting that something like a gap junction exists in

these cells, but the work has never been repeated. Innexins of gap

junctions have so far not been found in sponge genomes or

transcriptomes and dye coupling, usually an indication of gap-

junction-coupled cells, was not seen in dissociated cells of Haliclona

cf. permollis (Leys, 1995).

If electrical signalling occurred in cellular sponges, some faster

behavioural response to changes in the ionic medium would be

suspected, but this does not seem to be the case. Prosser (Prosser,

1967) showed that for sponges to contract, the water must have a

univalent ion (sodium could be replaced by potassium or lithium)

and a divalent cation (magnesium and calcium were usually both

required, although reduced contractions only occurred in the absence

of magnesium and strontium could replace calcium) (Fig. 3A).

Importantly, Prosser showed that contractions can occur at 10-fold

higher external potassium concentrations (100 mmol l

−1

), which

would normally depolarize cells, so he concluded it was unlikely

that action potentials were involved in contractions (Prosser, 1967).

Therefore, slower signalling pathways are expected, and these could

involve either small molecule transmitters (SMTs, including amino

acids, biogenic amines and gaseous molecules) or neuropeptides

(usually 3–40 amino acids long).

Although many SMTs are well known from plants and fungi, the

evolutionary origins of metazoan representatives of these molecules

are not entirely clear. Some of these molecules are found in sponge

transcriptomes and have been shown to function in the contraction

behaviour of sponges, but others do not seem to be produced by

sponges and may come from the sponges’ bacterial symbionts. For

example, there is evidence for the presence of metabotropic

glutamate and GABA receptors in the genomes of both

Amphimedon queenslandica and  Oscarella carmela, and

physiological experiments show that glutamate triggers contractions

and GABA inhibits contractions in the freshwater sponge (see

below). Despite an initial report that serotonin and dopamine

receptors were present in Amphimedon (Srivastava et al., 2010),

none have been found in transcriptomes of eight sponges or the

Amphimedon genome (Riesgo et al., 2014). Anti-serotonin

immunoreactivity was suggested for a sponge larva, but distribution

of the label was difficult to associate with any particular cell or cells,

and specificity of the antibody was not confirmed by western

blotting (Weyrer et al., 1999). Oddly, many papers report serotonin

or serotonin-like molecules (brominated cyclodipetides) in chemical

extracts from sponges (e.g. Hedner et al., 2006). As sponges are rich

sources of novel metabolites (Taylor et al., 2007), the majority of

which are produced by bacterial symbionts, we should consider

whether the major source of serotonin in sponges may actually be

bacterial symbionts.

Of the other SMTs (e.g. histamine, aspartate, ATP, cAMP GABA,

glutamate and the gaseous molecule NO) the function of glutamate

and GABA has been studied in most detail in the freshwater sponge

E. muelleri (Elliott and Leys, 2010). The sponge can be triggered to

‘sneeze’ by vigorous shaking (2–4 Hz) or by adding dilute Sumi

585

REVIEW

The Journal of Experimental Biology (2015) doi:10.1242/jeb.110817

---