Evidence for Vertical Transmission of Bacterial Symbionts from Adult to Embryo in the Caribbean Sponge Svenzea zeai

The Caribbean reef sponge Svenzea zeai was previously found to contain substantial quantities of unicellular photosynthetic and autotrophic microbes in its tissues, but the identities of these symbionts and their method of transfer from adult to progeny are largely unknown. In this study, both a 16S rRNA gene-based fingerprinting technique (denaturing gradient gel electrophoresis DGGE]) and clone library analysis were applied to compare the bacterial communities associated with adults and embryos of S. zeai to test the hypothesis of vertical transfer across generations. In addition, the same techniques were applied to the bacterial community from the seawater adjacent to adult sponges to test the hypothesis that water column bacteria could be transferred horizontally as sponge symbionts. Results of both DGGE and clone library analysis support the vertical transfer hypothesis in that the bacterial communities associated with sponge adults and embryos were highly similar to each other but completely different from those in the surrounding seawater. Sequencing of prominent DGGE bands and of clones from the libraries revealed that the bacterial communities associated with the sponge, whether adult or embryo, consisted of a large proportion of bacteria in the phyla Chloroflexi and Acidobacteria, while most of the sequences recovered from the community in the adjacent water column belonged to the class Alphaproteobacteria. Altogether, 21 monophyletic sequence clusters, comprising sequences from both sponge adults and embryos but not from the seawater, were identified. More than half of the sponge-derived sequences fell into these clusters. Comparison of sequences recovered in this study with those deposited in GenBank revealed that more than 75% of S. zeai-derived sequences were closely related to sequences derived from other sponge species, but none of the sequences recovered from the seawater column overlapped with those from adults or embryos of S. zeai. In conclusion, there is strong evidence that a dominant proportion of sponge-specific bacteria present in the tissues of S. zeai are maintained through vertical transfer during embryogenesis rather than through acquisition from the environment (horizontal transfer).Besides being the oldest metazoans, sponges are the simplest multicellular animals and possess a low degree of tissue differentiation and coordination (54). Sponges are sessile, filter-feeding organisms that may harbor within their tissues a remarkable array of microorganisms, including bacteria (19, 59, 64), archaea (41), zooxanthellae (22), diatoms (63), and fungi (35). In some cases, microbial consortia can make up to 40 to 60% of the sponge tissue volume (21, 61) and exceed a density of 10⁹ microbial cells per ml of sponge tissue (62), which is several orders of magnitude higher than that found in seawater. Apart from being a source of food (43), bacterial symbionts may participate in the acquisition and transfer of nutrients inside sponges (67, 68), the recycling of insoluble protein (69), the stabilization of the sponge skeleton (44), and the processing of metabolic waste (4, 65). Many antimicrobial compounds have been isolated from sponge bacterial symbionts (24, 47, 53), suggesting the involvement of symbiotic bacteria in sponge chemical defenses. In some cases, bacterial symbionts have been found to be the source of bioactive compounds that were isolated from sponges, which has opened up new research directions in marine natural product chemistry, biotechnology, and pharmaceutical development (18, 23, 40).Based on immunological evidence from the 1980s (66), sponge-bacterium symbioses are thought to have originated in the Precambrian, when bacteria evolved to form a single clade of sponge-specific bacteria that were distinct from isolates found in the surrounding seawater. Since then, many studies have similarly documented a high level of consistency and specificity in sponge-bacterium associations (20, 27, 59). Nevertheless, questions remain about the acquisition and maintenance of symbionts in host sponges. In general, the following two hypotheses have been proposed: (i) a recently metamorphosed sponge selectively retains specific groups of bacteria from the diverse pool of bacteria present in the water column as it begins filter feeding (horizontal transfer) or (ii) specific bacterial strains are transmitted by the maternal sponge to developing embryos and are already present in the metamorphosing sponge (vertical transfer) (58). The first hypothesis requires some recognition of specific microbes by the sponge, perhaps through an innate immune system (36) or other means to distinguish symbiont strains from food bacteria (70).Vertical transfer of bacterial symbionts in sponges was first proposed by Lévi and Porte (29), who demonstrated the presence of bacteria inside the larvae of the sponge Oscarella lobularis. Later, in 1976, Lévi and Lévi (30) studied the transmission of bacteria in the sponge Chondrosia reniformis via sponge oocytes. Since then, vertical transmission of bacterial symbionts via eggs or larvae has been documented for several sponge species, including Tethya citrina (15), Geodia cydonium (50), Stelletta grubii (49), Hippospongia sp. (25), Spongia sp. (25), Halisarca dujardini (10), and Corticium candelabrum (8). However, all of these studies employed transmission and scanning electron microscopy and could only examine the presence of bacteria in maternal sponges, oocytes, or larvae at the morphological level, with no determination of microbial identity. With advances in molecular techniques, Enticknap et al. (9) were the first to report the successful isolation of an alphaproteobacterial symbiont, strain NW001, from both the adult sponge Mycale laxissima and its larvae. They also did a preliminary denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial community in seawater and compared that with the community in the sponge larval sample. However, such a comparison was not extended to the sponge adult, and no solid conclusion can be drawn for the horizontal transfer mechanism of sponge symbionts. More recently, Sharp et al. (52) used fluorescence in situ hybridization (FISH) and clone library techniques to demonstrate the presence of proteobacteria, actinobacteria, and a clade of sponge-associated bacteria in the embryos and mesohyl of the tropical sponge Corticium sp. By clone library and DGGE analyses, Schmitt et al. (48a) identified 28 vertical-transmission clusters in five different Caribbean sponge species and demonstrated that the complex sponge adult microbial community was collectively transmitted through reproductive stages. While these recent studies support the vertical transfer hypothesis, they did not fully address the identities of microbes in the water column surrounding the sponges, which is key to determining whether horizontal transfer may also take place.The Caribbean reef sponge Pseudaxinella zeai was reclassified into a new genus, Svenzea (Demospongiae, Halichondria, Dictyonellidae), in 2002 because it has an unusual skeleton arrangement consisting mainly of short stout styles that are arranged in an isodictyal reticulation (2). It is a viviparous sponge that produces the largest embryos (>1 mm in diameter) and larvae (6 mm long) recorded for the phylum Porifera (45). Svenzea zeai has also been classified as a bacteriosponge because it contains substantial amounts of unicellular photosynthetic and autotrophic microbial symbionts in its tissues (2, 45). Although bacteria were observed in the embryos and larvae of this sponge based on transmission electron microscopy studies (45), neither the direct linkage between the maternal sponge and the propagules nor the identity of the microbial symbionts had been established.In this study, our objective was to examine vertical versus horizontal transfer of bacterial symbionts in Svenzea zeai. This was achieved by comparing the bacterial community profiles of the adults and embryos of the sponge by use of a combination of molecular techniques, including DGGE and clone library analysis. More than one technique was employed to compensate for deficiencies of each technique in revealing bacterial community structure. Additionally, we used the same techniques to examine the bacterial community in the seawater that surrounded the sponge to determine whether horizontal transfer was evident.