Genome analysis of Elysia chlorotica Egg DNA provides no evidence for horizontal gene transfer into the germ line of this Kleptoplastic Mollusc

Abstract

The sea slug Elysia chlorotica offers a unique opportunity to study the evolution of a novel function (photosynthesis) in a complex multicellular host. Elysia chlorotica harvests plastids (absent of nuclei) from its heterokont algal prey, Vaucheria litorea. The "stolen" plastids are maintained for several months in cells of the digestive tract and are essential for animal development. The basis of long-term maintenance of photosynthesis in this sea slug was thought to be explained by extensive horizontal gene transfer (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. Early studies of target genes and proteins supported the HGT hypothesis, but more recent genome-wide data provide conflicting results. Here, we generated significant genome data from the E. chlorotica germ line (egg DNA) and from V. litorea to test the HGT hypothesis. Our comprehensive analyses fail to provide evidence for alga-derived HGT into the germ line of the sea slug. Polymerase chain reaction analyses of genomic DNA and cDNA from different individual E. chlorotica suggest, however, that algal nuclear genes (or gene fragments) are present in the adult slug. We suggest that these nucleic acids may derive from and/or reside in extrachromosomal DNAs that are made available to the animal through contact with the alga. These data resolve a long-standing issue and suggest that HGT is not the primary reason underlying long-term maintenance of photosynthesis in E. chlorotica. Therefore, sea slug photosynthesis is sustained in as yet unexplained ways that do not appear to endanger the animal germ line through the introduction of dozens of foreign genes.

Keywords: Elysia chlorotica; Vaucheria litorea; genomics; horizontal gene transfer; kleptoplasty; symbiosis.

Fig. 1.

BLASTx results using assembled Vaucheria litorea cDNA contigs to query a comprehensive local genome database. This database consists of 16.9 million protein sequences derived from RefSeq v.51 plus sequenced eukaryote (e.g., fungal, metazoan, viridiplant, and stramenopile) genomes from the Joint Genome Institute (http://www.jgi.doe.gov/, last accessed May 16, 2013) and six-frame translated eukaryote EST sequences obtained from the NCBI dbEST (http://www.ncbi.nlm.nih.gov/dbEST/, last accessed May 16, 2013).

Fig. 2.

Comparative gernomics approach used to test for the presence of alga-derived genes (i.e., HGT candidates) in the germ line of Elysia chlorotica. (A) Algal transcriptome generated in this study. (B) Sea slug egg genomic data generated in this study. (C) Cummulative library of HGT candidates from past studies. Results of the various comparisons are illustrated and listed in detail in the indicated supplementary tables, Supplementary Material online. No algal or photosynthetic genes were detected in any of the queries performed with the E. chlorotica genomic reads.

Fig. 3.

PCR analysis showing the presence or absence of genes (A and B) or transcripts (C and D) of commonly identified HGT candidates in individual Elysia chlorotica collected from the wild (A and C) or 6-month-old laboratory-reared animals (B and D). Identical colored boxes represent the same individual being tested in panels A and C, and in panels B and D. Targeted genes are listed in table 1.

Fig. 4.

Percentage of animals (n = 3) showing the presence of genes (left) or transcripts (right) encoding 11 putative HGT candidates (divided into functional categories) and two control genes (actin and spermadine synthase). Tested animals were starved for 3 months (3MS) and then allowed to feed on Vaucheria litorea for 1 h (0HPF) and subsequently starved for 1 h (1HPF) or 24 h (24HPF). Controls indicate samples that had combined algal and animal nucleic acids as templates. Targeted genes are listed in table 1.