Bio-foam enhances larval retention in a free-spawning marine tunicate

Abstract

Here we report a mechanism that reduces dispersal of early developing stages and larvae in a free-spawning intertidal and shallow subtidal tunicate, Pyura praeputialis (Heller 1878), in the Bay of Antofagasta, Chile. The spawning of gametes by the tunicate into the naturally turbulent aerated seawater decreases their surface tension and induces the formation of a bio-foam. Water collected from foamy intertidal pools and tide channels showed a high concentration of P. praeputialis early developing stages and tadpole larvae in the foam. Because gametes are synchronically spawned for external fertilization and larvae settle near adults, our results suggest that this bio-foam increases fertilization success and effective settlement of their short-lived larvae in the vicinity of the adults spawning the gametes. This mechanism reinforces published evidence suggesting that local retention of intertidal and inshore marine invertebrate larvae may be more common than previously thought, offering, for instance, new perspectives for the design and networking of marine protected and management areas.

It is well known that surfactants can induce the formation of bio-foams that can ensure egg retention and larval development in organisms such as tropical leptodactylid frogs (1–5) and African and Asian freshwater labyrinth fishes (6). However, such egg-larval retentive mechanisms or “foam-nests” have not been reported for marine organisms. In Chile, massive populations of the intertidal and shallow subtidal tunicate Pyura praeputialis form beds almost exclusively along a 60–70 km stretch of the rocky shoreline inside the Bay of Antofagasta (7). This tunicate is an alien species in Chile, and its absence at locations more than 3–5 km outside of the Bay of Antofagasta, and along the Chilean coast, suggests the existence of unknown mechanisms preventing its dispersal (8) and maintaining high settlement inside the Antofagasta tunicate beds. In December 2004 and January 2005, midway through the ebb tides, we observed massive co-spawning of both eggs and sperm suspension from P. praeputialis into naturally turbulent aerated intertidal seawater (Fig. 1A) and the subsequent formation of localized and conspicuous masses of foams. These bio-foam masses persisted for several hours on top of rocky shores and floating in tidal channels, near where they were spawned, because of onshore winds (Fig. 1).

Fig. 1.

Photographic record of P. praeputialis spawning and field sampling. (A) Synchronous release of gametes from intertidal P. praeputialis during exposure to air. (B) Sperm and egg suspensions retained on the surface of intertidal specimens. (C) Thick foam (≈2 m height) covering the surface of tide channels. (D) Sampling of sea water from the surface in tide channels. ES, egg suspensions; SS, sperm suspensions.

Results and Discussion

To investigate the role of the bio-foam associated with P. praeputialis on the retention of embryos and tadpole larvae of the species, seawater samples were collected (December 10 and 12, 2004; January 14 and 15, 2005) by using a plankton bilge-pump equipped with a 50-μm elbow end and calibrated with a domestic flow meter (9). The pump was used to collect water samples at different time intervals through the tide cycle from the sea surface just above or near the intertidal rocky shores in Bay of Antofagasta that are dominated by P. praeputialis. Each sampling lasted for 10 min, filtering an average of 140 liters (±25 SE, n = 15). The pump, operated with a 12-V battery, was attached to a PVC float system that restricted sampling to the upper 10 cm of the water column. Water samples were fixed with formaldehyde and stored in labeled plastic bottles for further observations in the laboratory. In December, samples were collected over two alternate days and within each day in tide channels with foam. In January, samples were collected over two consecutive days and within each day, alternating between tidal channels with and without foam. Only samples from foamy waters showed the presence of embryos and tadpoles larvae of P. praeputialis (Fig. 2). In tide channels with foam, developing stages of P. praeputialis were found on the flood tide (Fig. 2). A few hours later, on the ebb tide, we detected the presence of tadpole larvae of P. praeputialis in the same channels (Fig. 2A). During the ebb tides, when most intertidal P. praeputialis are exposed to air, we observed synchronous spawning of both sperm and eggs suspensions from hundreds of tunicates (Fig. 1 A and B). This was followed by the formation of thick foam in the water surface (Fig. 1 C and D).

Fig. 2.

Abundance of developing stages and tadpole larvae of P. praeputialis per 10 liters of water collected from tide channels at different times of a tide cycle. (A) Symbols give the average number and standard error of developing embryos (circles) and tadpole larvae (squares) found in water samples collected during the day from three tide channels with (filled symbols) and without (open symbols) foam over two alternate days. (B) Symbols depict the average number and standard error of developing embryos (squares) and tadpole larvae (circles) found at diurnal hours in three water samples collected from tide channels with (filled symbols) and without (open symbols) foam along two consecutive days. Dashed lines indicate tidal cycles. Asterisks indicate direct observations of gamete spawns in the field.

To assess the potential role of retention associated with the foam, groups of 50 small plastic ping pong balls filled with seawater were placed in tide channels with and without foam midway through a sequential flood and ebb tide on December 17, 2004. After 2.5 h, all balls placed in the channels without foam were carried offshore by the currents. In contrast, in the same period 50% (flood) and 60% (ebb) of the balls remained in the foamy channels.

Foam nests enhance fertilization success and egg and larval retention in freshwater nest-building fishes (10) and in both aquatic and terrestrial frogs (5). In frogs, the production of bio-foams is associated with the production of “ranaspumins” (the surfactant properties of aqueous protein mixtures from the nest of the tropical frog Physalaemus pustulosus) by females (5), and those foams show active surfactant properties at about the same levels as we report for the tunicate bio-foam. One of the earliest records of coastal bio-foam associated their formation and stabilization with protein debris and alginates derived from damaged organism and algae (11). Thereafter, coastal bio-foams have been mainly associated with exceptionally high cell concentrations of the foam-forming colonial alga Phaeocystis spp. and kelp mucilage under specific wind and wave conditions (12–14). However, formation of coastal bio-foam has also been associated with detritus or fecal matter in wavy environments (15). Regardless of their source, coastal bio-foams (i.e., shore-cast foam or foam lines) have been described as enclosing marine metazoan fauna of several taxa and larvae (i.e., polychaete, mussel, and crustacean) (16, 17), and as a food resource for consumers (11, 16).

Hence, we conclude that the highly active surfactant bio-foam produced by P. praeputialis gametes, when spawned into turbulent aerated seawater, and the high number of tunicate developing embryos and larvae linked to foamy tide channels represent a new type of the global phenomenon of coastal bio-foams and a mechanism enhancing external fertilization and local retention of larvae in rocky shore environments. We suggest that in Antofagasta, the described bio-foam also enhances local tunicate settlement, because tadpole larvae of P. praeputialis are in the plankton for ≈2 h (18), less than the persistence of the bio-foam associated with P. praeputialis. Furthermore, this may be one of the mechanisms responsible for the as yet unexplained restricted distribution of this free-spawning species inside the Antofagasta Bay, as well as for its high settlement success on adult conspecifics inside the bay (19). Additionally, preliminary field observations in the intertidal rocky shores of Las Cruces and Pelancura (central Chile) have shown that a similar bio-foam production also occurs concurrently with massive gamete spawning of the sunstar Heliaster helianthus and for the chiton Acanthopleura echinata. This suggests that an active surfactant bio-foam retention mechanism, as described here, may be a more common phenomenon among intertidal or near-shore benthic free-spawning marine invertebrates, enhancing fertilization success and local rocky-shore retention of embryos and larvae with short planktonic periods. In the same vein, recent information indicates that various mechanisms [i.e., upwelling shadows, odor cues, and tidal fronts (7, 20–23)] linked to the retention of coastal marine fish and invertebrate larvae may be much more common than previously thought. This suggests, for example, that perhaps small no-take zones, or marine protected or management areas, may be indeed effective, and more desirable, for enhancement and recruitment of overfished or critical species; particularly if the areas are numerous and closed enough to allow a stepping-stone like process to operate between them (24, 25), and if the target species present short larval periods. Although the potential benefits of bio-foams in terms of increasing fertilization success could be quite general in marine free spawner invertebrates, their effects on the retention of larvae showing long planktonic stages need to be further investigated.

Material and Methods

In the laboratory, we tested the surfactant activity of P. praeputialis gonadal tissues. Male and female gonadic tissues were dissected from 15 adults under a stereomicroscope, stored separately, and frozen for chemical analyses. Tissues were defrosted, and samples were collected with a pipette to determine total protein concentrations by the Bradford method, monitoring the adsorption at 595 nm of the Coomassie brilliant blue-G250 bind to the proteins (26). For female gonads, 28.7 mg·ml⁻¹ concentration values were obtained; and for male gonads, 18.8 mg·ml⁻¹ concentration values were obtained. The interfacial tensions were measured at 298 K by the Wilhelmy plate method, using a Data Physics DCAT11 interfacial tensiometer. Water purified by using the Milli-Q system (Millipore) up a to resistivity of 18.22 MΩ·cm and with a surface tension value of >72.0 mN·m⁻¹ was used for dilution. The protein concentration for the analysis ranged between 0.10 and 410 μg·ml⁻¹. BSA (in phosphate buffer, pH 7) and male and female P. praeputialis gonad supernatant were used. Female and male gonadic extracts showed a strong reduction of water surface tension. At total protein concentrations of 18–24 μg·ml⁻¹, the surface tension drops sharply from pure water values, ≈72 mN·m⁻¹ to 66 mN·m⁻¹ with BSA and to ≈57 mN·m⁻¹ with male and female gonad tissues (Fig. 3). Around this protein concentration level, BSA reduces surface tension by ≈10%, whereas male and female gonads reduce surface tension by ≈21%.

Fig. 3.

Comparison of surfactant properties (tension) as a function of the protein concentration for BSA (open circles) and male gonad tissue (open squares) and female gonad tissue (filled triangles) of P. praeputialis. Error bars show 2 SD (n = 3).