FISH labeling reveals a horizontally transferred algal (Vaucheria litorea) nuclear gene on a sea slug (Elysia chlorotica) chromosome

2014-12-01 00:05:14

The Biological Bulletin;1 DEC 2014; Vol. 227: No. 3: 300-312

Julie A. Schwartz, Nicholas E. Curtis, Sidney K. Pierce


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.


Kleptoplasty, the process of phagocytosis, sequestration, and utilization of algal chloroplasts by certain digestive cells in some species of herbivorous sea slugs (Sacoglossa), is a well-known phenomenon that has been studied by many investigators for 50 years or more. The symbiotic plastids, kleptoplasts, can photosynthesize inside the slug cell for varying periods, from hours to months, depending upon the slug species, providing at least some source of energy for the animal. The longer-lived associations between the captured plastids and the slug involve various behavioral, morphological, and biochemical adaptations to maintain the chloroplasts in the absence of the mechanisms of organelle renewal that are present in the algal cell.

Of particular interest is the growing evidence that Elysia chlorotica, a slug species with one of the longest (9 mon or more) maintained associations with chloroplasts from its algal food, Vaucheria litorea, has somehow acquired functional algal nuclear genes. The products of these transferred genes help with the long-term maintenance of plastid function within the slug cell. Chloroplast reproduction has never been found in E. chlorotica (or any other sacoglossan), and the larval stages feed on unicellular algae. So, once metamorphosis to the adult form occurs, each generation of adult slugs must take up chloroplasts anew. Inside the adult E. chlorotica digestive cells, chloroplast proteins are synthesized for months after plastid uptake, and many of those are nuclear-encoded in the slug cell. Evidence for the presence of algal nuclear genes coding for chloroplast proteins in DNA from both E. chlorotica adults and larvae has been reported from polymerase chain reaction (PCR) experiments as well as real time PCR (qRT-PCR) and transcriptome sequencing of RNA from E. chlorotica adults. Therefore, all of these data agree that several dozen
algal nuclear genes have been transferred from the alga to slug cells, have been integrated into the slug cell biology, are transmitted to the next generation of slugs, and then are actively transcribed and translated in the animals. However, genome sequencing of E. chlorotica “egg ribbons,” which are actually unhatched larvae, failed to find any algal sequences. This negative result
was used to dismiss all of the previous positive results, including those with larvae, that demonstrated horizontal gene transfer between V. litorea and E. chlorotica, although “stress[ed]” that their results did not “prove” the absence of algal genes in the slug genome. Instead, alternative hypotheses were proposed to explain the mechanism of long-term chloroplast maintenance in E.
chlorotica without a gene transfer event incorporated into the germ line, including the possibility of extrachromosomal DNA fragments taken up during feeding and missed in the adult sequencing efforts. However, the unhatched embryos do not have chloroplasts and have not fed, so the source of the PCR-identified algal sequences in the larval DNA found in earlier studies cannot be from feeding. Furthermore, if extrachromosomal DNA were the source of the transferred algal genes in the adults, it would require the uptake of the correct DNA fragments by every cell in every slug in every generation.

Since a negative result from genomic data will always be subject at least to “not enough data” and “not in the database” criticisms, we have used a different approach to test the E. chlorotica genome for the presence of algal genes. Instead of just more genome sequencing, we used fluorescence in situ hybridization (FISH) and fluorescent microscopy to test chromosomes from pre-hatched E. chlorotica larvae (which have never been exposed to any algae) for the presence of algal sequences. We found that a gene probe made with the coding sequence of a V. litorea nuclear gene, prk, bound to one of the chromosomes in the unhatched larva of E. chlorotica, confirming the positive gene transfer results found by most of the previous studies.

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