Photosynthesis in some sacoglossan sea slugs offers a unique model to study the possibility of horizontal gene transfer (HGT) between multicellular predator and prey. Sacoglossan mollusks ingest and actively maintain chloroplasts that they acquire from large coenocytic green algae and keep them for up to several months (Wägele et al. 2011). These kleptoplastidic associations vary greatly in terms of specificity of the animal towards its algal prey and in retention time and functionality of the captured plastids (e.g., Rumpho et al. 2011, Klochkova et al. 2013). The basis for long-term maintenance of photosynthesis in these sea slugs has often been explained by extensive HGT from the nucleus of the alga to the animal nucleus, followed by expression of algal genes in the gut to provide essential plastid-destined proteins (Bhattacharya et al. 2013).
Sacoglossan mollusk Placida dendritica Alder et Hancock is bonded avidly to its specific algal food Bryopsis spp. (Klochkova et al. 2010). When it develops from a veliger larva to a small animal, at final stage of metamorphosis it attaches to and consumes only Bryopsis plants for the rest of its life cycle. In north Atlantic waters, P. dendritica is commonly found associated with Codium fragile (Suringar) Hariot (Evertsen and Johnsen 2009), but most Korean samples were collected on Bryopsis spp. In laboratory culture with mixed diet algae, the animals consumed only Bryopsis spp. and less than 10% of them switched to Codium minus (Schmidt) Silva or Derbesia tenuissima (Moris et De Notaris) P. L. Crouan et H. M. Crouan, even when no Bryopsis was available (Klochkova et al. 2010). Chloroplasts were observed in the digestive tract of P. dendritica and were found to be functional for a short time (Klochkova et al. 2010).
In this study, we present initial results from the comparative analysis of transcriptomes of P. dendritica and its food alga B. plumosa that suggest the maintenance of photosynthesis in the sea slug is not directly related with the horizontally transferred genes from algae. However, possible case of putative HGT was found, such as an actin gene from P. dendritica.
Adult animals of P. dendritica (Fig. 1) were collected from Wando, southern coast of Korea (34°19′37.32″ N, 120°48′43.55″ E). Collected sea slugs were washed with filtered artificial seawater and kept in marine IMR medium at 15℃ with 12 : 12 h L : D cycle and 15 μmol m-2 s-1 light intensity and without food.
For transcriptome analysis, sea slugs were kept without any food algae (i.e., starved) for 28 days and the Petri dish with culture medium was changed every day. Egg ribbons were harvested from one week after starvation, when no chloroplasts remained in the digestive tract of the sea slugs, because defecation stopped by that time and their body color was turning yellowish by each passing day. The harvested eggs were rinsed with 3% H2O2, frozen in liquid nitrogen and kept in deep freezer (−70℃) until use. Other sacoglossan sea slugs, Elysia atroviridis Baba and Elysia nigrocapitata Baba, were collected from the same locality. The animals were maintained using same method as for P. dendritica.
All algae (Appendix 1) were cultured in marine IMR medium at 20℃ with 12 : 12 h L : D cycle and 15 μmol m-2 s-1 light intensity.
Total RNA from B. plumosa and P. dendritica was isolated using Trizol (MRC Inc., Cincinnati, OH, USA) according to manufacturer’s protocol. Thirty animals of P. dendritica, which had been starved for 28 days, were used. Isolated RNA was quantified spectrophotomertically (260 and 280 nm). mRNA was purified using Oligotex (Qiagen, Valencia, CA, USA) following manufacturer’s instructions. Double-strand cDNA was synthesized using Just cDNA Double-stranded cDNA Synthesis Kit (Agilent Technologies, Palo Alto, CA, USA) following manufacturer’s instructions. The cDNA was then sent to Macrogen (http://www.macrogen.com/eng/) for 454 pyrosequencing. The library preparation, GS-FLX titanium sequencing, assembly and annotation of sequencing data were carried out by Macrogen (Appendix 2). To analyze the sequence data a web-based pipeline program for expressed sequence tag (EST) data analysis was established (http://genebank2.kongju.ac.kr).
DNA was purified from algal samples from different localities (Appendix 1) and the eggs of sea slugs using Intron i-genomic plant DNA extraction mini kit or CTAB DNA extraction mini kit (Intron Biotechnology, Seoul, Korea) following the manufacturer’s instructions. Isolated DNA was diluted to 10 ng μL-1 of concentration and directly used to PCR reaction. Specific primers were designed based on pyrosequencing database (Appendix3). PCR was performed as follows: an initial denaturation at 95℃ for 4 min, followed by 35 cycles of amplification (denaturation at 94℃ for 30 s, annealing at 55℃ for 40 s, and extension at 72℃ for 1.5 min) with a final extension at 72℃ for 10 min.
Transcriptome of B. plumosa was compared with that of P. dendritica by local blast program based on nucleotide sequence (BlastN) from BioEdit ver. 7.0 (Ibis Therapeutics, Carlsbad, CA, USA). All the contigs and singletons of the two species were compared and a table of similar genes was generated. The contigs and singleton sequences smaller than 200 bp were removed from the data set. BlastN parameters were set to expectation value >1.0e-60 and identity >85% using BLOSUM62 matrix. Genes were selected by keyword searches from the final spreadsheets obtained from the above annotation process. Functional annotation was used to obtain matches with the following terms: photosynthesis, chlorophyll, light harvesting, intracellular transport, metabolic processes (carbohydrates, lipids, and proteins), organelle organization and biogenesis. Additional homology searches were conducted by comparing our translated EST database directly with the comprehensive chloroplast protein database of Arabidopsis thaliana (plastid protein database: http://www.plprot.ethz.ch and AT-Chloro database: http://www.grenoble.prabi.fr/at_chloro) with a cut-off E-value of e-50 (Kleffmann et al. 2004). The BioEdit sequence alignment editor program (ver. 7.2.3) was used for sequence homology analysis. An EST database of Dictyostelium discoideum Raper obtained from NCBI EST database (dbEST ID: 13952321) was compared with the assembly results of B. plumosa using the method described above. As there were no significant matches at the BlastN parameters above, the parameters were lowered to expectation value >1.0e-20 and identity >75% using BLOSUM62 matrix.
The actin sequences examined in this study were aligned with the actin sequences from GenBank using MacClade 4.08. The nucleotide alignment contained 65 sequences and was trimmed to 778 nucleotide comparable positions without third codon position using Paup*v. b10. Maximum likelihood analysis was performed using RAxML 7.0.4 with rapid bootstrapping option and 1,000 replicates under GTR + I + Γ model. Tree was visualized and graphic versions were exported using FigTree v1.4.0. New actin sequences generated in this study have been deposited in NCBI under the accession numbers listed in Appendix 4. Accession numbers for the sequences from NCBI used to construct phylogenetic tree are listed in Appendix 5.
Functional annotation on P. dendritica transcriptome showed no putative gene related to the following terms: light and dark reaction of photosynthesis, chlorophyll assimilation, and light harvesting complex. Additional homology searches comparing P. dendritica transcriptome with the comprehensive chloroplast protein database of A. thaliana (Kleffmann et al. 2004) showed no significant match, except for some ribosomal genes with a cut-off Evalue of e-50.
Comparison of two large-scale ESTs databases of B. plumosa and P. dendritica showed few candidates of putative HGT except an actin homologue (Table 1). Nine actin homologues were isolated from P. dendritica EST database and three from B. plumosa (Table 2). One actin homologue from P. dendritica, designated as PdActin04, showed 98.9% identity in DNA sequence with the complementary gene from B. plumosa, BpActin03, while all the other genes, including other actin homologues, ribosomal proteins, and tubulin genes of the two species showed much lower similarity (≤86%) (Table 1). Full sequence of PdActin04 was obtained from genomic PCR using the egg cells of P. dendritica. Highly conserved homologues (93-99% of DNA sequence identity) of this gene were found in eight other ulvophyceaen algae (Appendix 6). However, PdActin04 homologue was not found in the eggs of other related sacoglossan species (Elysia atroviridis, E. nigrocapitata), which also feed on Bryopsis spp. The sequence difference between BpActin03 and PdActin04 was similar to that between species of Bryopsis. Most DNA substitution among BpActin03 homologues of ulvophyceaen algae were synonymous; the translated amino acid sequences were almost identical (>99.7%) to each other. It is noteworthy that three DNA substitutions occurring in PdActin04 were not synonymous and not observed in any other green algae (Fig. 2).
Phylogenetic analysis showed incongruence between the actin gene and known organismal phylogenies of the animals and algae (Fig. 3). Surprisingly, all BpActin03 homologues were grouped within a branch of Amoebozoa, while other actin genes from the two species were grouped appropriately in the Archaeplastida and Opisthokonta (Fig. 3). To check if PdActin04 homologues are from simultaneous contamination of some Amoebozoa species, the transcriptomes of P. dendritica and B. plumosa were compared with that of an amoeba D. discoideum. No genes from two EST databases showed significant homology with that of D. discoideum (Table 3).
Our results did not show any photosynthesis-related genes in P. dendritica transcriptome, which suggest that HGT may not be the primary reason underlying the maintenance of photosynthesis in this mollusk. To become an active nuclear-encoded functional chloroplast protein and return to the plastid, the transferred gene must be assimilated into the host nuclear genome, acquire a transit sequence for targeting the protein to the organelle and be transcribed and processed back into the chloroplast (Bhattacharya et al. 2013). It would be less surprising that a common cytosolic gene like actin which may not require all the steps described above has been successfully transferred and transcribed between two intimately associated organisms. The specialized feeding and use of algal organelles by the sacoglossan mollusks also support the possibility of HGT among them.
The gold standard for identifying HGT with confidence is phylogenetic incongruence and this occurs if there is strong conflict between the phylogenies of the gene and of the organism (Keeling and Palmer 2008). The incongruence between the gene and known organismal phylogenies of the herbivore and algae did not support that PdActin04 has been horizontally transferred from its food algae. PdActin04 and all of its homologues found in green algae were nested in a branch of the Amoebozoa phylogeny (Fig. 3). It is possible that BpActin03 homologues are from a common amoeba contaminating all algal strains as well as the sea slugs simultaneously. If the contamination was from laboratory culture, all BpActin03 homologues should have the same sequence.
However, DNA sequences of nine BpActin03 homologues were clearly different among species. It means there must be nine different amoebas specifically contaminating algal strains, as well as the sea slug. It is hard to believe that each algal strain, collected from different localities of the world in different times, carry a specific amoeba and never mixed during years of laboratory culture. Most of all, the sequence difference among the homologues reflected the phylogenetic distance among ulvophyceaen algae; species closely related showed less sequence difference (Appendix 6). DNA sequence homology among BpActin03 homologues ranged 93-99%.
Direct comparison of large EST databases enabled us to avoid a long-lasting concern with HGT studies, that of contamination of targeted genes. If the materials used for building EST database of P. dendritica were contaminated with B. plumosa the transcriptomes of the two species would share many genes in common. Simultaneous contamination of the two EST databases by a common amoeba would also reveal more genes in common between B. plumosa and P. dendritica, not just one actin gene. Although all these evidences indicate that BpActin03 homologues are not from a contaminating amoeba, the questions about how and when this actin gene transferred to green algal lineage still remains.
The intimate physical interaction between herbivore and food algae may lead to horizontal transfer of certain genes. The short-term kleptoplastidy occurring in Placida dendritica does not seem to be based on any genetic incorporation from the food algae, Bryopsis spp. An interesting actin lineage was found and gene was isolated as a candidate of putative HGT between them, but the incongruence between the gene and known organismal phylogenies did not support the possibility of HGT. Highly conserved actin gene lineage found in this study may be useful in interpreting the evolutionary relationship among higher level of taxa.
