Solar-Powered Sea Slugs

ORIGINAL: U Maine | Nature

Imagine being able to turn on solar-powered cells whenever food became limiting in your environment to exploit the sun’s energy to produce chemical energy. Further imagine the advantage of being mobile and camouflaged with a green, rippling leaf appearance in a sea filled with predators in search of soft-bodied creatures. The sacoglossan mollusc Elysia chlorotica (Gould) possesses all of these traits. It is a shell-less, green “walking leaf” that will feed on algae when they are available, stealing the chloroplasts, and using them for solar-power when food is scarce.

ORIGINAL: Nature

The slug pictured to the right, Elysia chlorotica, is a symbiont thief.

Elysia chlorotica eats the alga Vaucheria litorea but does not digest it. The slug cuts open algal filaments and sucks out the contents, transferring the living chloroplasts to its own tissue. Chloroplasts are organisms that have lived symbiotically within plant cells for many millions of years. They harness energy from the sun, which they give to the plant or alga cell they live within. Most animals digest the chloroplasts entirely when they eat plants, but not Elysia. By keeping the chloroplasts intact and transferring them to its own tissue, Elysia allows them to continue photosynthesizing, producing energy for the slug. The slug can then live for months without eating as long as sunlight is available, and can maintain the same chloroplasts for its entire adult life. This is an extremely unique relationship between an animal and plant symbionts.

Many other animals form associations with photosynthetic organisms. Corals such as the one depicted below have a symbiosis with multiple single-celled organisms called zooxanthellae. This is a multiple-level symbiosis because corals house the entire chloroplast-containing zooxanthellae cells within their tissue. This is different from Elysia chlorotica, who has cut out the middleman — instead of incorporating entire cells, it only retains the chloroplasts.
The photograph of Elysia chowing down was taken by Nicholas E. Curtis and Ray Martinez. The second photograph of Elysia is courtesy of Mary S. Tyler, and was the cover of PNAS when this paper was published. The lower picture is the coral Porites as photographed by Casey Dunn.

You can watch two amazing videos of the slugs in action, here and here, both of which were included in the PNAS paper.
--Freya Goetz

These sea slugs feed by slicing or puncturing siphonaceous algal cells and sucking out the cell contents. All of the contents, including the algal nucleus, are discarded except the chloroplasts which are engulfed phagocytotically into the digestive cells. (The figure to the right shows the "stolen" chloroplasts, or kleptoplasts, within the cells of the slug's digestive tract.)

By distributing the “photosynthetic factories“ throughout their extensively branched digestive system just one cell layer beneath the epidermis, the sea slugs not only blend into the green algal bed (the figure to the right shows the sea slug on strands of the alga Vaucheria litorea ), they also capture light energy to fuel photoautotrophic CO2 fixation. In some cases, the resulting carbon products can totally sustain the sea slugs for several months in the absence of an algal food source and serve as precursors for synthesis of chemical defense compounds and the copious mucus which bathes and protects the sea slugs.

Symbiosis
Symbiotic associations between organisms, even of different kingdoms, is not that unusual. However, in almost all cases they represent an association between two intact, free-living organisms, both of which have retained their complete cellular genetic make-up. Such associations are typically intercellular and, if intracellular, the symbiont is frequently isolated from the host’s cytosol by sequestration in a vacuole or host-provided membrane. What makes the sea slug/algal chloroplast symbiosis so remarkable is that the symbiont is a “naked,” foreign organelle sustained intracellularly in direct contact with the host sea slug cytosol and the symbiont remains functional for several months despite the absence of any algal nucleo-cytosolic influence.

There is some disagreement on whether an association between an organism and an isolated organelle such as a chloroplast constitutes symbiosis since the symbiont (chloroplast) is not a free-living organism. The term symbiosis was first defined as, “unlike organisms” living together. “Unlike organisms” came to mean different species and symbiosis changed to reflect, “prolonged physical associations without respect to outcome.” In the early 1900's, the Russian scientist K.S. Mereschkovsky proposed that chloroplasts originated from blue-green algae (cyanobacteria), a process he named symbiogenesis or “the origin of evolutionary novelty via symbiosis.”

In 1975, Robert Trench defined intracellular symbiosis as, “the coexistence of at least two genomes of divergent evolutionary origins occupying the same cytoplasmic environment.”

In her book on “Symbiotic Interactions,” Angela Douglas (1994) emphasizes that symbiosis is not dependent on mutual benefit to the partners, rather that at least one of the partners acquires a new metabolic property. Considering both Trench’s and Douglas’ definitions, we conclude that the intracellular association of algal chloroplasts with molluscan cells can be considered a unique symbiotic association. The chloroplast represents a symbiont genome and the host mollusc acquires a new metabolic capability, photosynthesis. Still, others prefer to use the term kleptoplasty or “something borrowed” to describe the chloroplast symbiosis. Regardless of definition or term used, today it is universally recognized that great biological novelty and diversity come from symbiotic associations and symbiosis is a widespread biological phenomenon.

PRIMARY, SECONDARY AND TERTIARY ENDOSYMBIOSIS

The endosymbiotic events leading to a solar-powered sea slug.

The endosymbiont theory traces the origin of the chloroplast to a free-living cyanobacterium that was engulfed by a eukaryote giving rise to the primary lineages of glaucophytes, red algae (rhodophytes), and the green plants and algae (the viridiplantae or streptophytes and chlorophytes, respectively).

Subsequently, secondary endosymbiosis, the uptake of a eukaryotic alga (green or red lineage) by another heterotrophic eukaryotic host, gave rise to a diverse group of secondary or complex algae, including the heterokont Vaucheria litorea.

Tertiary symbiosis is most commonly associated with dinoflagellates and the replacement of their endosymbiont with a new secondary endosymbiont. Here, we propose that the engulfment of secondary chloroplasts by a sea slug also represents a tertiary endosymbiotic association.

Kleptoplasty by Elysia chlorotica.
History
The first reports of "green bodies" within molluscs were made in 1904 by Brüel. However, it was not until the mid 1960's that these "green bodies" were studied and seen to be functional chloroplasts. From this time on, there has been much interest in the phenomenon by which sea slugs find, acquire, and maintain chloroplasts as an essential component of their life-cycle.

Kleptoplasty (“stealing” of chloroplasts) by Elysia chlorotica is remarkable for at least three reasons.

1. First, the “symbiont” in this case is not another autonomous organism with an intact genome, but rather a “naked,” foreign organelle (chloroplast).
2. Second, the symbiont is housed intracellularly and not sequestered between cells or within a vacuolar membrane.
3. Third, and perhaps most remarkable, the semi-autonomous kleptoplasts remain functional for as long as ten months within the foreign host cytosol despite the absence of any of its own algal nucleo-cytosolic components.

This type of long-term activity by isolated plastids is unprecedented and astounding considering that chloroplasts are derived from once free-living cyanobacteria and have lost the majority of their genes. Hence, they are dependent on their own nucleo-cytosol for protein synthesis, targeting, and regulation of just about every function of the organelle. In turn, chloroplasts are fragile organelles and very sensitive to physical and chemical changes in their environment, including osmotic stress. Evidently, the advantages of photoautotrophy have provided a strong selective pressure for the evolution of this association.

Whether the establishment of this symbiotic association is fueled by photoautotrophy pressure to sustain energy production for the sea slug when food is scarce or the need for camouflage protection when facing life without a protective shell in a predatory environment, or both, the association has not progressed to a hereditary one where the plastids are passed from one generation of slugs to the next. Instead, the association must be established anew each year and is required for the slug to develop into a mature adult sea slug, at least in laboratory experiments.

Establishment of the Kleptoplastic Association
We have now succeeded in culturing Elysia chlorotica in the laboratory including establishing the symbiotic or kleptoplastic association with Vaucheria litorea chloroplasts. Adult E. chloroticaproduce eggs, devoid of plastids, typically in late spring and planktonic veligers hatch within 4 to 5 d and then spend about 5 d feeding on unicellular algae (Rhodomonas or Isochrysis in the lab). When subsequently provided with filaments of V. litorea, metamorphosis of the veligers into juvenile sea slugs occurs within 1 to 2 d.

The endosymbiosis is established when young juvenile sea slugs grasp and then puncture the siphonaceous algal cells and suck out the cell contents. All of the algal contents are “discarded” except the chloroplasts, which are engulfed phagocytotically into the digestive cells. The captured chloroplasts fill the growing, extensively branched digestive tubules that lie just one cell layer beneath the epidermis.

Over the next several months the sea slugs may continue to feed onVaucheria if it is available and/or sustain themselves by photoautotrophic CO2 fixation using their newly acquired chloroplasts.

In laboratory culture, the sea slugs are kept apart from the algae, thus sustaining themselves totally by photosynthesis for up to ten months. Interestingly, death of the adults occurs almost synchronously late each spring in the lab and field (Pierce et al. 1999 Biol. Bulletin on virus).

The alga vaucheria
Vaucheria: Synonyms = water felt
Reproduction = asexual by fragmentation of filaments or zoospores and sexual by oogamy.

Vaucheria is a salt-water, yellow-green alga (Xanthophyte), in the Heterokont Kingdom.

Xanthophytes (yellow-green algae): >600 species, PS, fresh and marine waters, Chl a and c1 and c2 and NO fucoxanthin or Chl b. Cell wall, but not of cellulose or chitin. Food reserve = oil or fat, not starch typically. Haploid nucleus with outer membrane continuous with ctER. Sessile or free-living; motile flatellated unicells to colonies to siphonaceous (coenocytic) multinucleated filaments, to multicellular filaments.

Heterokont (=stramenopile = chromista)
description: Members of the Kingdom Chromista or Heterokontae exhibit the following characteristics: tubular mitochondria, motile cell with two different flagella. They vary from unicellular flagellates to large, siphonaceous filaments. They include diatoms, raphidiophytes, chrysophytes (golden algae), oomycota (water molds), phaeophytes (brown algae and kelps), haptophytes, silicoflagellata, and the xanthophyta (yellow-green algae including Vaucheria).

Vaucheria exhibits a siphonaceous morphology with a large vacuole and a thin layer of multinucleate cytoplasm containing numerous chloroplasts.

Chloroplasts

The chloroplasts ofVaucheria litorea have the standard four membranes surrounding them when seen within the algal cytoplasm. When isolated, they have only the standard double envelope.

Vaucheria litorea.
Taxonomy:

Kingdom Chromista (=Heterokontae)

Division (Phylum): Ochrophyta

Class: Xanthophyceae

Order: Vaucheriales

Family: Vaucheriaceae

Genus: Vaucheria (De Candolle, 1801)

Species: Vaucheria litorea Hofman ex C. Agardh, 1823

Culturing the alga vaucheria

The heterokont alga (= chromophytes [chl a and c], autotrophic stramenopiles), Vaucheria litorea, is maintained in culture in enriched quarter-strength (tolerates ¼ to full-strength sea water) Instant Ocean and a modified f/2 medium with natural lighting and daily manual swirling.

about elysia chlorotica

Found in saltwater marshes along the east coast of the US as far north as Nova Scotia to NC in the south and some times down to FL; waters of widely varying salinity, but usually brackish salt marshes. Our specimens are routinely collected from an intertidal marsh on Martha’s Vineyard Island, MA.

Synonyms = “leaves that crawl,” “solar-powered sea slugs,” “walking leaf,” “eastern emerald elysia,”

Reproduction = hermaphroditic, but self-fertilization is not common, shed fertilized eggs

Size = 2 to 3 cm common; some up to 6cm