Algal Sources of Sequestered Chloroplasts in the Sacoglossan Sea Slug Elysia crispata Vary by Location and Ecotype.

Sacoglossan sea slugs feed by suctorially consuming siphonaceous green algae. Most sacoglossan species are feeding specialists, but the Caribbean coral reef-dwelling Elysia crispata is polyphagous and sequesters chloroplasts from multiple algal species into cells lining its digestive diverticulum for use in photosynthesis. We have used sequences of the chloroplast-encoded rbcL gene to compare the chloroplast donor algae in five populations of E. crispata from various Caribbean locations. We found that E. crispata utilizes more algal species than was previously known, including some algae previously not reported as present in the region. In addition, slugs from each location had unique chloroplast arrays with little overlap, except that all locations had slugs feeding on algae within the genus Bryopsis. This variation in diet between locations suggests that the slugs may be exhibiting local adaptation in their dietary choices, and it highlights ecological differences between the Caribbean-wide reef-dwelling ecotypes and the mangrove lagoon ecotypes found in the Florida Keys.

A draft genome assembly of the solar-powered sea slug Elysia chlorotica.

Elysia chlorotica, a sacoglossan sea slug found off the East Coast of the United States, is well-known for its ability to sequester chloroplasts from its algal prey and survive by photosynthesis for up to 12 months in the absence of food supply. Here we present a draft genome assembly of E. chlorotica that was generated using a hybrid assembly strategy with Illumina short reads and PacBio long reads. The genome assembly comprised 9,989 scaffolds, with a total length of 557 Mb and a scaffold N50 of 442 kb. BUSCO assessment indicated that 93.3% of the expected metazoan genes were completely present in the genome assembly. Annotation of the E. chlorotica genome assembly identified 176 Mb (32.6%) of repetitive sequences and a total of 24,980 protein-coding genes. We anticipate that the annotated draft genome assembly of the E. chlorotica sea slug will promote the investigation of sacoglossan genetics, evolution, and particularly, the genetic signatures accounting for the long-term functioning of algal chloroplasts in an animal.

A Preliminary Molecular and Phylogenetic Analysis of the Genome of a Novel Endogenous Retrovirus in the Sea Slug Elysia chlorotica.

An endogenous retrovirus that is present in the sea slug Elysia chlorotica is expressed in all individuals at the end of the annual life cycle. But the precise role of the virus, if any, in slug senescence or death is unknown. We have determined the genomic sequence of the virus and performed a phylogenetic analysis of the data. The 6060-base pair genome of the virus possesses a reverse transcriptase-domain-containing protein that shows similarity to retrotransposon sequences found in Aplysia californica and Strongylocentrotus purpuratus. However, nucleotide BLAST analysis of the whole genome resulted in hits to only a few portions of the genome, indicating that the Elysia chlorotica retrovirus is novel, has not been previously sequenced, and does not have great genetic similarity to other known viral species. When more invertebrate retroviral genomes are examined, a more precise phylogenetic placement of the Elysia chlorotica retrovirus can be determined.

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.

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.

Cell biology of the chloroplast symbiosis in sacoglossan sea slugs.

Chloroplasts removed from their species of origin may survive for various periods and even photosynthesize in foreign cells. One of the best studied and impressively long, naturally occurring examples of chloroplast persistence, and function inside foreign cells are the algal chloroplasts taken up by specialized cells of certain sacoglossan sea slugs, a phenomenon called chloroplast symbiosis or kleptoplasty. Among sacoglossan species, kleptoplastic associations vary widely in length and function, with some animals immediately digesting chloroplasts, while others maintain functional plastids for over 10 months. Kleptoplasty is a complex process in long-term associations, and research on this topic has focused on a variety of aspects including plastid uptake and digestive physiology of the sea slugs, the longevity and maintenance of symbiotic associations, biochemical interactions between captured algal plastids and slug cells, and the role of horizontal gene transfers between the sea slug and algal food sources. Although the biochemistry underlying chloroplast symbiosis has been extensively examined in only a few slug species, it is obvious that the mechanisms vary from species to species. In this chapter, we examine those mechanisms from early discoveries to the most current research.

Foraging behavior under starvation conditions is altered via photosynthesis by the marine gastropod, Elysia clarki.

It has been well documented that nutritional state can influence the foraging behavior of animals. However, photosynthetic animals, those capable of both heterotrophy and symbiotic photosynthesis, may have a delayed behavioral response due to their ability to photosynthesize. To test this hypothesis we subjected groups of the kleptoplastic sea slug, Elysia clarki, to a gradient of starvation treatments of 4, 8, and 12 weeks plus a satiated control. Compared to the control group, slugs starved 8 and 12 weeks displayed a significant increase in the proportion of slugs feeding and a significant decrease in photosynthetic capability, as measured in maximum quantum yield and [chl a]. The 4 week group, however, showed no significant difference in feeding behavior or in the metrics of photosynthesis compared to the control. This suggests that photosynthesis in E. clarki, thought to be linked to horizontally-transferred algal genes, delays a behavioral response to starvation. This is the first demonstration of a link between photosynthetic capability in an animal and a modification of foraging behavior under conditions of starvation.

PHYLOGENETIC ANALYSIS OF THE LARGE SUBUNIT RUBISCO GENE SUPPORTS THE EXCLUSION OF AVRAINVILLEA AND CLADOCEPHALUS FROM THE UDOTEACEAE (BRYOPSIDALES, CHLOROPHYTA)(1).

The placement of Avrainvillea and Cladocephalus in the family Udoteaceae (order Bryopsidales) has been questioned on the basis of nuclear, plastid, and other ultrastructural characteristics unique to these genera. Bayesian analysis of the chloroplast-encoded LSU RUBISCO (rbcL) gene showed that the Udoteaceae is paraphyletic. Cladocephalus luteofuscus (P. Crouan et H. Crouan) Børgesen, Avrainvillea nigricans f. floridana D. Littler et Littler, and A. mazei G. Murray et Boodle form a clade with the freshwater alga Dichotomosiphon tuberosus (A. Braun ex Kütz.) A. Ernst that is basal to a clade that includes other members of the Udoteaceae, the Halimedaceae, and the Caulerpaceae. The noncalcified species Boodleopsis pusilla (Collins) W. R. Taylor, A. B. Joly et Bernat. groups with species of the calcified Udoteacean genera Penicillus, Rhipocephalus, Udotea, and Halimeda.

Microscopic, biochemical, and molecular characteristics of the Chilean Blob and a comparison with the remains of other sea monsters: nothing but whales.

We have employed electron microscopic, biochemical, and molecular techniques to clarify the species of origin of the "Chilean Blob," the remains of a large sea creature that beached on the Chilean coast in July 2003. Electron microscopy revealed that the remains are largely composed of an acellular, fibrous network reminiscent of the collagen fiber network in whale blubber. Amino acid analyses of an acid hydrolysate indicated that the fibers are composed of 31% glycine residues and also contain hydroxyproline and hydroxylysine, all diagnostic of collagen. Using primers designed to the mitochondrial gene nad2, an 800-bp product of the polymerase chain reaction (PCR) was amplified from DNA that had been purified from the carcass. The DNA sequence of the PCR product was 100% identical to nad2 of sperm whale (Physeter catadon). These results unequivocally demonstrate that the Chilean Blob is the almost completely decomposed remains of the blubber layer of a sperm whale. This identification is the same as those we have obtained before from other relics such as the so-called giant octopus of St. Augustine (Florida), the Tasmanian West Coast Monster, two Bermuda Blobs, and the Nantucket Blob. It is clear now that all of these blobs of popular and cryptozoological interest are, in fact, the decomposed remains of large cetaceans.

PROLINE BETAINE: A UNIQUE OSMOLYTE IN AN EXTREMELY EURYHALINE OSMOCONFORMER.

The extremely euryhaline mollusc, Elysia chiorotica, does not utilize intracellular free amino acids for cell volume regulation during osmotic stress. Instead, Elysia utilizes an osmolyte previously unknown from animals, proline betaine. Although proline betaine occurs in some plants and Elysia forms a symbiosis with an algae, the proline betaine in Elysia seems to be a product of the animal.

Ultrastructure of the heart of the marine mussel, Geukensia demissa.

The structure of the heart of Geukensia demissa, a common object of physiological and biochemical investigation, is described by scanning, transmission and freeze-fracture electron microscopy. A single-cell epithelial layer covers the ventricle, but an endothelium is lacking. Myofibers are small (6-7 µm diam.), mononucleate, and tapered. Glycogen is concentrated peripherally. Mitochondria are particularly concentrated under the sarcolemma, near the ends of the nucleus, and in rows between bundles of myofilaments. The myofilaments (6-8nm thin, 30-35 nm thick filament diam.) are loosely arranged into sarcomeres (2-4 µm) by Z bodies. Many of these Z bodies interconnect, and some anchor to the sarcolemma forming attachment plaques. Cells are joined by intercalated discs consisting of fascia adherentes, spot desmosomes, and gap junctions. The gap junctions include intramembrane particles. T tubules are absent. The sarcolemma is coupled to the junctional sarcoplasmic reticulum (JSR) over 357ndash;40% of the cell surface. Tubules extend from the JSR deep into and throughout the cell as an irregularly dispersed network. The SR occupies 1% of the cell volume. A few, small (0.1-1.0 µm) unmyelinated nerves are present, but no neuromuscular junctions were seen. The auricles have fewer and smaller myocytes than the ventricle. The auricles also contain podocytes with pedicels having 20-35 nm slits and containing sieve-like projections. The morphology of the Geukensia heart is similar to that of other bivalves.