Julie A. Schwartz

Transcriptomic evidence for the expression of horizontally transferred algal nuclear genes in the photosynthetic sea slug, Elysia chlorotica.

Analysis of the transcriptome of the kleptoplastic sea slug, Elysia chlorotica, has revealed the presence of at least 101 chloroplast-encoded gene sequences and 111 transcripts matching 52 nuclear-encoded genes from the chloroplast donor, Vaucheria litorea. These data clearly show that the symbiotic chloroplasts are translationally active and, of even more interest, that a variety of functional algal genes have been transferred into the slug genome, as has been suggested by earlier indirect experiments. Both the chloroplast- and nuclear-encoded sequences were rare within the E. chlorotica transcriptome, suggesting that their copy numbers and synthesis rates are low, and required both a large amount of sequence data and native algal sequences to find. These results show that the symbiotic chloroplasts residing inside the host molluscan cell are maintained by an interaction of both organellar and host biochemistry directed by the presence of transferred genes.

Using Algal Transcriptome Sequences to Identify Transferred Genes in the Sea Slug, Elysia chlorotica

The first molecular evidence of horizontal gene transfer between multicellular eukaryotes was our discovery of the presence of three Vaucheria litorea nuclear-encoded genes [fucoxanthin chlorophyll a/c-binding protein (fcp) and light-harvesting complex 1 and 2 (Lhcv1 and 2)] in the genomic DNA of the sea slug, Elysia chlorotica, which are used to support the chloroplast endosymbiosis in the slug. These genes are translated and transcribed in the host cell, and vertically transmitted to subsequent generations of the host species. In order to provide a database of native V. litorea sequences to facilitate the search for additional transferred genes between these two species, we have partially sequenced and annotated the transcriptome of V. litorea, using 454 Life Science’s next generation pyrosequencing technology. Preliminary analysis of the sequence data has led to the discovery of six additional algal nuclear genes in E. chlorotica cDNA and genomic DNA, which encode enzymes in the chlorophyll synthesis pathway as well as additional light-harvesting and metabolic enzymes. Furthermore, we confirm the recent discovery of the Calvin-Benson cycle gene, prk.

Chlorophyll a synthesis by an animal using transferred algal nuclear genes

Chlorophyll synthesis is an ongoing requirement for photosynthesis and a ubiquitous, diagnostic characteristic of plants and algae amongst eukaryotes. However, we have discovered that chlorophyll a (Chla) is synthesized in the symbiotic chloroplasts of the sea slug, Elysia chlorotica, for at least 6 months after the slugs have been deprived of the algal source of the plastids, Vaucheria litorea. In addition, using transcriptome analysis and PCR with genomic DNA, we found 4 expressed genes for nuclear-encoded enzymes of the Chla synthesis pathway that have been horizontally transferred from the alga to the genomic DNA of the sea slug. These findings demonstrate the first discovery of Chla production in an animal using transferred nuclear genes from its algal food.

FISH Labeling Reveals a Horizontally Transferred Algal (Vaucheria litorea) Nuclear Gene on a Sea Slug (Elysia chlorotica) Chromosome

The horizontal transfer of functional nuclear genes, coding for both chloroplast proteins and chlorophyll synthesis, from the food alga Vaucheria litorea to the sea slug Elysia chlorotica has been demonstrated by pharmacological, polymerase chain reaction (PCR), real time PCR (qRT-PCR), and transcriptome sequencing experiments. However, partial genomic sequencing of E. chlorotica larvae failed to find evidence for gene transfer. Here, we have used fluorescent in situ hybridization to localize an algal nuclear gene, prk, found in both larval and adult slug DNA by PCR and in adult RNA by transcriptome sequencing and RT-PCR. The prk probe hybridized with a metaphase chromosome in slug larvae, confirming gene transfer between alga and slug.

The Intracellular, Functional Chloroplasts in Adult Sea Slugs ( Elysia crispata ) Come from Several Algal Species, and are Also Different from those in Juvenile Slugs.

The sacoglossan sea slug, Elysia crispata, sequesters chloroplasts from its algal food source within specialized cells lining the digestive diverticulum. These stolen chloroplasts photosynthesize within the slug cell cytoplasm as long as four months--one of the longest kleptoplastic associations known [1]. While many other sacoglossan species feed on and sequester chloroplasts from only one species of algae, adult E. crispata sequester plastids from three different species of algae; Penicillus capitatus, Halimeda incrassata, and Halimeda monile [2]. We have now done feeding experiments testing the ability of newlymetamorphosed, juvenile E. crispata, raised from egg masses in the lab, to sequester chloroplasts from multiple algal species using a large range of potential algal food sources. Surprisingly, juvenile E. crispata fed on different algal species (Bryopsis plumosa and Derbesia tenuissima) from those utilized for sources of symbiotic plastids in the adults. Transmission electron microscopy (TEM) verified that the B. plumosa and D. tenuissima chloroplasts were sequestered intracellularly in the juvenile slugs. In addition, juvenile E. crispata fed exclusively on B. plumosa could be grown to adult size, and, as adults, they would switch to feeding on Penicillus capitatus if presented with it. Since the fine structure of B. plumosa and P. capitatus chloroplasts are easily distinguishable, TEM indicated that both types of chloroplasts are sequestered simultaneously inside the same cell in animals fed on both species of algae (Fig. 1). Finally, a newly discovered population of E. crispata which lives in an area where only B. plumosa is present showed the presence of B. plumosa chloroplasts sequestered in adult slug digestive cells using TEM analysis and using molecular markers. Adult slugs fed on B. plumosa in the lab maintained chloroplasts for approximately as long as the field-collected animals. These results indicate that E. crispata not only eats several species of algae, but also is capable of maintaining symbiotic plastids concurrently from those species for long periods.