Sacoglossan Sea Slugs Research Articles

The kleptoplastic sea slug Elysia clarki prolongs photosynthesis by synthesizing chlorophyll a and b

Several species of kleptoplastic, sacoglossan sea slug photosynthesize using chloroplasts sequestered inside their digestive cells from algal food sources. However, sequestered chloroplasts alone are not sufficient for months-long, continuous photosynthesis and maintenance of the chloroplasts in absence of the algal nucleus. Some type of plastid maintenance mechanism must be present to help sustain photosynthetic activity in the long term kleptoplastic species, such as Elysia clarki. We demonstrate that E. clarki starved for 2 weeks are able to synthesize chlorophylls, but that slugs starved for 14 weeks no longer synthesize chlorophyll. The subsidence of chlorophyll synthesis is coincident with the cessation of photosynthesis by the starved slugs, but it is not yet known if the cessation of pigment synthesis is the cause or some other aspect of plastid degradation produces a loss of synthetic ability.

Effects of photosynthesis on the survival and weight retention of two kleptoplastic sacoglossan opisthobranchs

Many sacoglossan sea slugs utilize chloroplasts ingested from food algae for photosynthesis (functional kleptoplasty), and the extent and duration of kleptoplast retention differs greatly among sacoglossan species. Although most recent studies focus on the genetic, microscopic, or physiological mechanisms responsible for this unique phenomenon, its effects on the life history traits of sacoglossans have not been fully explored. To study the effects of light conditions on survival and weight retention, adult individuals of two sacoglossan species, Elysia trisinuata and Plakobranchus ocellatus (‘black type'), were reared under light conditions (a 14-hour light: 10-hour dark photoperiod with an irradiance level of 28 µmol m−2s−1) or complete darkness for 21 days. There was no significant difference in the survival rate between the light and dark treatments for E. trisinuata, and its wet weight relative to the initial weight was smaller in the light than in the dark. However, both the survival and relative weights were greater in the light than dark for P. ocellatus. Based on the fluorescent yield measurement using pulse-amplitude-modulated fluorometry, the retention duration of functional chloroplasts was longer (>17 days) for P. ocellatus than E. trisinuata (<4 days). These results indicate that P. ocellatus benefits from photosynthesis for survival and growth, whereas E. trisinuata does not under starved conditions. This interspecific difference is likely related to the period of functional chloroplast retention.

What makes a chloroplast? Reconstructing the establishment of photosynthetic symbioses

Earth is populated by an extraordinary diversity of photosynthetic eukaryotes. Many eukaryotic lineages contain chloroplasts, obtained through the endosymbiosis of a wide range of photosynthetic prokaryotes or eukaryotes, and a wide variety of otherwise non-photosynthetic species form transient associations with photosynthetic symbionts. Chloroplast lineages are likely to be derived from pre-existing transient symbioses, but it is as yet poorly understood what steps are required for the establishment of permanent chloroplasts from photosynthetic symbionts. In the past decade, several species that contain relatively recently acquired chloroplasts, such as the rhizarian Paulinella chromatophora, and non-photosynthetic taxa that maintain photosynthetic symbionts, such as the sacoglossan sea slug Elysia, the ciliate Myrionecta rubra and the dinoflagellate Dinophysis, have emerged as potential model organisms in the study of chloroplast establishment. In this Commentary, we compare recent molecular insights into the maintenance of chloroplasts and photosynthetic symbionts from these lineages, and others that might represent the early stages of chloroplast establishment. We emphasise the importance in the establishment of chloroplasts of gene transfer events that minimise oxidative stress acting on the symbiont. We conclude by assessing whether chloroplast establishment is facilitated in some lineages by a mosaic of genes, derived from multiple symbiotic associations, encoded in the host nucleus.

Amphi-Atlantic distributions and cryptic species in Sacoglossan sea slugs

Nearly 13% of opisthobranch gastropods in the Atlantic Ocean are regarded as amphi-Atlantic (i.e. species occurring on both eastern and western coastlines of the Atlantic realm). This assumption has been broadly based on morpho-anatomical similarities and has rarely been tested within a molecular phylogenetic framework. Among opisthobranchs, the order Sacoglossa is renowned for its difficult and confusing species taxonomy, particularly in some genera like Elysia, where several cases of cryptic speciation have been documented. Moreover, the width of the Atlantic realm poses a serious challenge to the idea of trans-Atlantic dispersal, particularly in nonplanktotrophic species. The species Bosellia mimetica, Elysia timida and Thuridilla picta are three sacoglossan opisthobranchs with amphiAtlantic status, but doubts have been raised about the conspecificity of eastern and western populations. Here we used methods to test molecular phylogenetic whether the Atlantic Ocean constitutes a barrier for dispersal and hence whether eastern and western populations belong to the same species. We used the criteria of divergence and reciprocal monophyly, supported by concordant genealogically independent genetic markers, to define species. Maximum-likelihood and Bayesian analyses of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes and nuclear gene Histone-3 (H3) were used to produce phylogenetic trees. We conclude that the eastern Atlantic species B. mimetica is not amphi-Atlantic, although the H3 gene did not recover reciprocal monophyly between western and eastern populations, perhaps in a result of incomplete lineage-sorting. Elysia timida is the only species showing an amphi-Atlantic distribution, but possibly due to recent introduction in the tropical western Atlantic. Thuridilla picta is restricted to the western Atlantic and therefore not amphi-Atlantic. Populations of ‘T. picta’ inhabiting the Macaronesian archipelagos of Azores and Madeira are a colour form of the eastern Atlantic T. hopei. Our results highlight the difficulty surrounding systematics of Sacoglossa sea slugs and provide further evidence that cryptic speciation is common in the group.

Transcriptomic Evidence That Longevity of Acquired Plastids in the Photosynthetic Slugs Elysia timida and Plakobranchus ocellatus Does Not Entail Lateral Transfer of Algal Nuclear Genes

Sacoglossan sea slugs are unique in the animal kingdom in that they sequester and maintain active plastids that they acquire from the siphonaceous algae upon which they feed, making the animals photosynthetic. Although most sacoglossan species digest their freshly ingested plastids within hours, four species from the family Plakobranchidae retain their stolen plastids (kleptoplasts) in a photosynthetically active state on timescales of weeks to months. The molecular basis of plastid maintenance within the cytosol of digestive gland cells in these photosynthetic metazoans is yet unknown but is widely thought to involve gene transfer from the algal food source to the slugs based upon previous investigations of single genes. Indeed, normal plastid development requires hundreds of nuclear-encoded proteins, with protein turnover in photosystem II in particular known to be rapid under various conditions. Moreover, only algal plastids, not the algal nuclei, are sequestered by the animals during feeding. If algal nuclear genes are transferred to the animal either during feeding or in the germ line, and if they are expressed, then they should be readily detectable with deep-sequencing methods. We have sequenced expressed mRNAs from actively photosynthesizing, starved individuals of two photosynthetic sea slug species, Plakobranchus ocellatus Van Hasselt, 1824 and Elysia timida Risso, 1818. We find that nuclear-encoded, algal-derived genes specific to photosynthetic function are expressed neither in P. ocellatus nor in E. timida. Despite their dramatic plastid longevity, these photosynthetic sacoglossan slugs do not express genes acquired from algal nuclei in order to maintain plastid function.

Ultrastructure of sequestered chloroplasts in sacoglossan gastropods with differing abilities for plastid uptake and maintenance

Abstract. Many sacoglossan sea slugs incorporate intact, functional chloroplasts from their algal food sources into specialized cells lining the digestive diverticulum. The chloroplasts in adults of Elysia clarki are photosynthetically functional for many months. Members of this species feed on algae in the Ulvophyceae, including species of Penicillus and Bryopsis. However, other sacoglossans (Elysia patina, Elysia rufescens, and Placida kingstoni) use similar algal food sources as do adults of E. clarki, but are unable to maintain the chloroplasts for more than a week, with individuals of P. kingstoni apparently being unable to maintain chloroplasts for >24 h. We have examined chloroplast sequestering cells of these species looking for morphological differences that may help explain the variation in chloroplast sequestration and maintenance among them. Our results indicate that P. kingstoni does not actively sequester chloroplasts at all, digesting them instead. However, the plastid sequestering mechanisms of individuals of E. patina and E. rufescens are similar to those of E. clarki, and the degradation of chloroplasts by specimens of E. patina is ultrastructurally similar to the same process in E. clarki, although chloroplast degradation occurs much more slowly in individuals of E. clarki. Our results suggest that species‐level differences in the digestive capability of the phagosomes involved in the uptake of chloroplasts account for variation in the length of these kleptoplastic associations.

Molecular Phylogeny of the Sacoglossa, With a Discussion of Gain and Loss of Kleptoplasty in the Evolution of the Group

Sacoglossan sea slugs retain the chloroplasts from food algae in their cells (kleptoplasty) and obtain the photosynthetic products, but the capability of kleptoplasty differs among species. One evolutionary hypothesis for kleptoplasty is that the family Volvatellidae is the ancestral sacoglossan lineage in which kleptoplasty arose, but this is based on ambiguous phylogenetic relationships. To test this hypothesis, we analyzed sacoglossan phylogeny for concatenated sequences (approximately 4000 bp) of nuclear (18S rRNA) and mitochondrial (CO1, trnV, and 16S rRNA) genes of 18 sacoglossan species, using neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian methods. The resultant phylogenetic trees clearly revealed monophyly of the order Sacoglossa (including the debatable genus Cylindrobulla), which contained two sister clades: the shelled sacoglossan group (Oxynoacea + Cylindrobulla) and the non-shelled group (Plakobranchacea). The family Volvatellidae was in a derived position within the Oxynoacea. The most parsimonious rearrangement of the character states of kleptoplasty on the phylogenetic tree suggested that (1) kleptoplasty was acquired at the basal position of the Sacoglossa (including Cylindrobulla) as non-functional kleptoplasty, (2) functional kleptoplasty was gained in the Plakobranchacea clade, and (3) non-functional kleptoplasty was lost in the Volvatellidae lineage in the Oxynoacea clade.

Slugs’ last meals: molecular identification of sequestered chloroplasts from different algal origins in Sacoglossa (Opisthobranchia, Gastropoda)

Some sacoglossan sea slugs have become famous for their unique capability to extract and incorporate functional chloroplasts from algal food organisms (mainly Ulvophyceae) into their gut cells. The functional incorporation of the so-called kleptoplasts allows the slugs to rely on photosynthetic products for weeks to months, enabling them to survive long periods of food shortage over most of their life-span. The algal food spectrum providing kleptoplasts as temporary, non-inherited endosymbionts appears to vary among sacoglossan slugs, but detailed knowledge is sketchy or unavailable. Accurate identification of algal donor species, which provide the chloroplasts for long-term retention is of primary importance to elucidate the biochemical mechanisms allowing long-term functionality of the captured chloroplast in the foreign animal cell environment. Whereas some sacoglossans forage on a variety of algal species, (e.g. Elysia crispata and E.viridis) others are more selective. Hence, characterizing the range of functional sacoglossan-chloroplast associations in nature is a prerequisite to understand the basis of this enigmatic endosymbiosis. Here, we present a suitable chloroplast gene (tufA) as a marker, which allows identification of the respective algal kleptoplast donor taxa by analysing DNA from whole animals. This novel approach allows identification of donor algae on genus or even species level, thus providing evidence for the taxonomic range of food organisms. We report molecular evidence that chloroplasts from different algal sources are simultaneously incorporated in some species of Elysia. NeigborNet analyses for species assignments are preferred over tree reconstruction methods because the former allow more reliable statements on species identification via barcoding, or rather visualize alternative allocations not to be seen in the latter.

Kleptoplasts mediate nitrogen acquisition in the sea slug Elysia viridis

Kleptoplasty is a remarkable form of symbiosis whereby photosynthetically functional algal chloroplasts are retained by the host organism. Certain sacoglossan sea slugs form such associ- ations and some species such as Elysia viridis (Montagu, 1804) can survive for months without access to their food algae. We report evidence for light-dependent assimilation of ammonium, nitrite, and urea (but not nitrate) in E. viridis with functional kleptoplasts. N uptake was decreased in slugs with reduced chloroplast densities, and after exposure to inhibitors of glutamine synthetase and glutamate synthetase. Incorporation of 15 N-labeled substrates was traced into individual hydrolyzable amino acids, with highest incorporation in glutamine-glutamate. N assimilation by kleptoplasts in multi- cellular hosts constitutes an additional mechanism which may contribute to the unique and long-lived functionality of these symbioses.

The symbiotic chloroplasts in the sacoglossanElysia clarkiare from several algal species

Abstract.The sacoglossan sea slugElysia clarkifeeds on siphonaceous algae, and intracellularly sequesters chloroplasts, which actively photosynthesize for 4 months. We have determined the algal source of chloroplasts in adults ofE. clarkifrom the Florida Keys, using molecular techniques, feeding experiments, and electron microscopy. Our results clearly demonstrate that specimens ofE. clarkisequester chloroplasts from four different species of algae, representing two genera:Penicillus lamourouxii, P. capitatus, Halimeda incrassata, andH. monile.In addition, chloroplasts from more than one species of algae are sequestered simultaneously in the same digestive cell.

Newly metamorphosed Elysia clarki juveniles feed on and sequester chloroplasts from algal species different from those utilized by adult slugs

The adult, sacoglossan sea slug, Elysia clarki (Pierce et al. in Molluscan Res 26, 2006), sequesters functional chloroplasts in cells of the digestive diverticula from four species of macroalgae in the order Bryopsidales (Penicillus capitatus, Penicillus lamourouxii, Halimeda incrassata, and Halimeda monile). Feeding experiments were conducted in December, 2003, using individuals raised in the laboratory from egg masses laid by E. clarki adults which had been collected from Grassy Key, Florida, USA, and 29 species of macroalgae collected from the Florida Keys, Tampa Bay, Tarpon Springs, Florida, or Woods Hole, Massachusetts or obtained from the Culture Collection of Algae at the University of Texas at Austin, Texas. For the first 14-day post-metamorphosis, juveniles ate only the thin filamentous species, Bryopsis plumosa or Derbesia tenuissima. Transmission electron microscopy showed that the chloroplasts from both algae were sequestered intracellularly in juvenile slugs. Individuals offered any other macroalga, including the four species fed on by adults, did not feed on or incorporate any chloroplasts, and soon died. Juveniles switched from B. plumosa to P. capitatus at a length of ∼ 1.0 cm, and fixed for microscopy 14 days later had intact intracellular chloroplasts from both algae. Nucleotide sequences of the chloroplast gene, rbcL, from DNA extracted from E. clarki, collected from Vaca Key, Florida, were aligned with 22 other available macroalgal rbcL sequences indicating that these E. clarki adults had fed on three species of algae in the Bryopsidales including Bryopsis pennata, and TEM results partially confirmed this conclusion.

A morphological and molecular comparison between Elysia crispata and a new species of kleptoplastic sacoglossan sea slug (Gastropoda: Opisthobranchia) from the Florida Keys, USA

A comparison of Elysia (=Tridachia) crispata (Mörch, 1863) from the Virgin Islands with elysiid slugs from the mangrove swamps and canals in the Florida Keys that have previously been identified as E. crispata reveals many differences in habitat, gross and microscopic anatomy, food preferences of juveniles, sources of symbiotic chloroplasts and their localization within the digestive tubules, radular morphology, and nucleic acid sequences of two genes. The differences between the two groups of slugs are such that the Florida Keys animals are considered to represent a new species, Elysia clarki.

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.

Spermatocyte chromosomes and genome size in the sacoglossan slugStiliger felinus(Hutton 1882) (Gastropoda: Opisthobranchia) from New Zealand

Abstract A karyological investigation was carried out on the testis cells of the tidal pool sacoglossan sea slug Stiliger felinus from New Zealand. The haploid chromosome set was characterised by 14 elements where median‐, subterminal‐, and terminal‐centromere chromosomes were present. Using flow cytometric analysis, haploid genome size of the species was found to be 2.8 pg: the highest value encountered in the subclass Opisthobranchia. Stiliger felinus differs from other Stiligeridae in chromosome number and frequency of cross‐shaped bivalents in spermatocytal metaphase I plates.

A note on some sacoglossan penial styles (Gastropoda: Opisthobranchia)

A penial style is a hollow, cuticular extension of the vas deferens present in most sacoglossan sea-slugs and also in many eolids and some dorids. During copulation a style projects beyond the tip of the distended penis and is used to transfer sperm either by hypodermic injection, or by conducting the sperm to a bursa copulatrix. The flexible styles of bivalved gastropods are described for the first time and new details are given for some other sacoglossan styles. The evolution of penial styles and their uses in taxonomy are discussed.