Nudibranch and sponge samples were collected using a combination of SCUBA from vessels, shore-based SCUBA, and snorkel from locations around the islands of Moorea (17.5333°S, 149.8333°W) and Tahiti (17.6667°S, 149.4167°W), French Polynesia, between 2011 and 2013 (Table 1). In addition, a fragment of a Porites rus coral colony – harboring eggs and adults of the nudibranch Phestilla lugubris – was collected from the field and kept for one month in an aquarium (open water flow). Eggs, juveniles and adult nudibranchs were separated from their coral substratum. All specimens were preserved in 85% ETOH at 4 °C.

Whole genomic DNA was extracted from the ethanol-preserved tissue. A CTAB protocol, modified after Mieog et al. [19], was used. Specific genomic regions typically used as a reference for barcoding were amplified by PCR.

To confirm the taxonomic identification of the collected species, “universal” primers [20] were used to amplify by PCR a 658 bp DNA region of the cytochrome c oxidase subunit 1 gene (COI) from either the nudibranch or sponge mitochondrial genome. To detect symbionts, a close to 580 bp region of the Symbiodinium large ribosomal subunit RNA (28S rDNA) was amplified with Richter's universal primers [21]. Amplifications by PCR were performed following the manufacturer's protocol (Promega Corporation, Madison, WI). Amplicons were purified and sent for sequencing of both strands to Macrogen (www.macrogen.com). Complementary sequences were edited and assembled into contigs using Genious pro 5.5.6 software (Biomatters Ltd, New Zealand). Multiple sequence alignment was performed using MEGA5 software [22]. Gene sequences were deposited in GenBank.

The amplified sequences of the partial COI and 28S rDNA regions were compared with the sequences available in the public nucleotide databases at the National Center for Biotechnology Information (NCBI) by using its World Wide Web site (http://www.ncbi.nlm.nih.gov), and the BLAST-algorithm [23]. Publicly available sequences exhibiting the highest similarities with our data have been retained to construct the dinoflagellate tree. The phylogenic reconstruction was performed by the maximum-likelihood method with the nucleotide substitution model “General Time Reversible” using a discrete Gamma distribution (best model fitted) embedded in the MEGA5 software for dinoflagellate 28S data set. 28S rDNA sequences of species of the phylum Apicomplexa were chosen as outgroup for the dinoflagellate tree (Fig. 1).

Hill et al. [12] showed by cloning that excavating clionaid sponges from the Indo-Pacific region harbor Symbiodinium from clades A, C, and G, with the majority of sponge hosts harboring only one clade. This report presents the first evidence that a filtering sponge (OTU QM2538) harbors clade C Symbiodinium (KJ500000) (Fig. 1). To date it has only been frequently observed that L. herbacea, and other related sponges, host filamentous cyanobacterium Oscillatoria spongeliae [25]. The fragment (KJ500000) showed similarity to Symbiodinium C90 (JN558047) hosted by foraminifera Sorites sp. [26] Except for the fact that type C90 has previously been found in corals [7], relatively little is known about this association.

Concerning nudibranchs, this study is also the first to date to examine the presence of Symbiodinium in numerous species. The 28S rDNA dinoflagellate primers successfully amplified a 565 bp fragments in different species of nudibranch tested as well as in the coral Porites rus. Our results show for the first time that the nudibranch Aeolidiella alba harbors a clade B representative (KJ500001) with a high similarity to the subclade B1 found in the soft coral Plexaura kuna (JN558057). Clade B had never been found before in French Polynesia, but previous studies had only looked at hard corals [7, unpublished work of the CRIOBE]. It is noteworthy that Venn et al. revealed the abundance of Symbiodinium clades A and B in a tropical sea anemone [27]. Moreover, members of the genus Aeolidiella feed on anemones, which means that A. alba could have taken up clade B from their prey. It would therefore be interesting to find out more about the Symbiodinium associated with anemones of French Polynesia.

Finally, this study shows the first direct sequence comparison of the Symbiodinium found in a nudibranch and its coral substratum, except Ziegler et al. [28]. All life stages of the nudibranch Phestilla lugubris (KJ522463), from eggs, to juveniles and to adults (Fig. 1) were collected, and sequence analysis revealed a new 28S rRNA gene of a so far unknown subclade C (KJ499999). Not only was the same clade detected in eggs, juveniles and adults, but the sequence alignment also revealed a 100% identity along 522 bp. This significant result supports that P. lugubris is very likely to be a case of direct inheritance of the symbionts (vertical transfer). In addition, the associated invertebrate host species – the coral Porites rus – on which the predator P. lugubris feeds, was analyzed. Sequencing data revealed that both the nudibranch and the coral carry exactly the same clade of Symbiodinium (KJ499999) (Fig. 1). The novelty of this work comes from the fact that those two species had never been described as interacting together, and that all life stages of P. lugubris occur on P. rus. This argues in favor of the existence of a horizontal transfer of symbionts, from coral to adult nudibranchs, which in turn will transfer its symbionts to their eggs.

The size of the obtained PCR fragments did not always correspond to the expected 565 bp 28SrDNA size: in addition, another 700 bp amplicon was sometimes detected. The amplified 700 bp fragment (KJ499997) of the sponge Haliclona sp. (OTU QM4715) had a similar sequence (94%) to the free-living dinoflagellate Dinophysis lativelata (AB473665) [29] (Fig. 1). The sponge Phycopsis. sp. (OTU QM1640), which was expected to be Symbiodinium-free, was found to host species (KJ499996) with a 28S gene sequence similar to a genus of a new planktonic mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. isolated from coastal waters off Western Korea (AM408889) (Fig. 1). The latter has recently been described by Kang et al. [30].

The partial 28S DNA sequence (KJ500002) amplified from the nudibranch Flabellina sp. suggests that it could either be a new genus or a new species within the order Gymnodininales or genus Gymnodinium (Fig. 1).

Two possible hypotheses could explain these new observations: (i) Dinophysis and Gymnodinium are free-living and use host tissues as habitats to aggregate and reproduce or (ii), host tissues contain symbiotic lineages of Dinophysis and Gymnodinium. Histological analyses should shed more light on this. To date, only planktonic Dinophysis and Paragymnodinium are referenced. It is known that dinoflagellates Gymnodinium spp. and Pfisteria piscicida kleptoplastids (notably chloroplasts) are photosynthetically active for only a few days, while kleptoplastids in Dinophysis spp. can continue to function for two months [31] and therefore could be an alternative pathway to provide energy to the host.

Correlation between the clades present in various hosts from a single location reveals that clades A, C and D were found in corals from Moorea's lagoon [7]; clade B and C were found in the studied nudibranch and sponge specimens from Moorea's and/or Tahiti's lagoon (Table 1). Since nudibranchs can move from one location to another and release healthy symbionts to the environment [15], this study reveals their ability to act as potential reservoirs and vectors of viable Symbiodinium for the whole reef ecosystem. They may be able to provide the corals or anemones they feed on with Symbiodinium, by discharging them in their vicinity, allowing, for example, symbiont switching after a bleaching event or in case of changing environmental conditions. A more comprehensive set of data concerning the clades present, their uptake, turnover and seasonal flexibility in nudibranchs and sponges, together with their coral substrates or preys, will enable the assessment of their role in buffering the coral reefs’ response to climate change and ocean acidification.

To conclude, this work provides numerous new elements, which will serve to better characterize the nature of the relationships involving Symbiodinium and invertebrate host species. Other areas of interest should now be considered, such as the study of metabolic exchanges between host and symbionts: the rate and nature of nutrient exchanges has not yet been studied in sponges and nudibranchs. The extent to which they rely on the dinoflagellates for survival is still under investigation. Also, the question of the stability and specificity of the relationship needs to be studied more extensively. Indeed, it has been recently demonstrated in corals [7] that flexible hosts exhibit a higher sensitivity to environmental changes; they have a higher propensity to bleach and higher mortality rates. Hosts, which have a high degree of fidelity towards their symbionts, may be more environmentally resilient. Compared to corals, sponges and nudibranchs have alternate and quite efficient feeding mechanisms, which make the role of Symbiodinium clades less important in their acclimatization to changing environments. Further investigations on the sponges and nudibranchs’ ability to switch from one Symbiodinium clade to another linked to their fitness in a changing environment will determine if specific associations are required for survival.