Chemical defenses in Sacoglossan Opisthobranchs : Taxonomic trends and evolutive implications *

Sacoglossan sea slugs (Mollusca: Opisthobranchia) are one of the few groups of specialist herbivores in the marine environment. Sacoglossans feed suctorially on the cell sap of macroalgae, from which they “steal” chloroplasts (kleptoplasty) and deterrent substances (kleptochemistry), retaining intracellularly both host plastids and chemicals. The ingested chloroplasts continue to photosynthesize for periods ranging from a few hours or days up to 3 months in some species. Shelled, more primitive sacoglossans feed only on the siphonalean green algal genus Caulerpa, and they do not have functional kleptoplasty. The diet of Sacoglossans has radiated out from this ancestral food. Among the shell-less Plakobranchidae (=Elysiidae), the more primitive species feed on other siphonales (families Derbesiaceae, Caulerpaceae, Bryopsidaceae and Codiaceae) and fix carbon, while the more “advanced” species within the Plakobranchidae and Limapontioidae have a more broad dietary range. Most of these “advanced” species are unable to fix carbon because the chloroplasts of their food algae are mechanically disrupted during ingestion. Mesoherbivores are likely to be eaten if they live on palatable seaweeds, their cryptic coloration and form not always keeping them safe from predators. Sacoglossans prefer to live on and eat chemically defended seaweeds, and they use ingested algal chemicals as deterrents of potential predators. The most ancestral shelled sacoglossans (Oxynoidae) and some Plakobranchidae such as Elysia translucens, Thuridilla hopei and Bosellia mimetica have developed a diet-derived chemical defense mechanism. Oxynoids and Thuridilla hopei are able to biomodify the algal metabolites. However, the Plakobranchidae Elysia timida and E. viridis, together with Limapontioidea species, are characterized by their ability to de novo synthesize polypropionate metabolites. A whole analysis of kleptoplasty and chemical defenses in Sacoglossans may offer a better understanding of the ecology and evolution of these specialized opisthobranchs. In this paper we summarize some of the latest findings, related mainly to Mediterranean species, and offer a plausible evolutionary scenario based on the biological and chemical trends we can distinguish in them.

If you are looking for alternatives to Darwinism, don't waste your time on opisthobranchs.
Whatever the immediate future of the research on the consortia in which sacoglossans and other opisthobranchs are involved, it will presumably provide as many surprises and as much satisfaction as in the last few years.J.D. Ros and A. Marín (1991).
It has seemed to me convenient to introduce the term "kleptodefenses" to describe the defensive systems of any type which an opisthobranch obtains borrowed from another animal, from a plant or, even, from inert materials in the environment.J.D. Ros (1976).

INTRODUCTION
Most marine herbivores (fishes, sea urchins, amphipods, crabs, polychaetes and gastropods) are extreme feeding generalists.In contrast to marine herbivores, terrestrial insect herbivores have numerous feeding modes, such as sap sucking, leaf mining, and galling, that are largely unknown among marine herbivores (Hay and Steinberg, 1992).Seaweeds differ from terrestrial plants by the lack of phloem and xylem transport systems.The only group of seaweeds that have extensive nonseptate tubes of cytoplasm are the siphonous green seaweeds, or Siphonales.Sacoglossans (Mollusca: Opisthobranchia) are one of the few groups of specialized herbivores in the marine environment.These sea slugs feed suctorially on the cell sap of siphonous green algae and of some macroalgae with high cellular cytoplasm volume.Sacoglossans "steal" chloroplasts (kleptoplasty) and deterrent substances (kleptochemistry) from the food algae and retain both host plastids and chemicals intracellularly.The ingested chloroplasts continue to photosynthesize for periods ranging from a few hours or days up to 3 months in some species (Marín and Ros, 1989).Sacoglossans feed suctorially by piercing the algal cell wall with a specialized stylet-like tooth (Clark, 1992).The radula of sacoglossans is highly modified to sap sucking, with a continuous single row of teeth.There are two basic shapes of teeth in the Sacoglossa: the sabot-shaped teeth found in the genera Stiliger, Ercolania, Limapontia and Alderia, and the blade-shaped teeth which occur in all other genera (Fig. 1,top).The particular use of these teeth also varies from one genus to another, the mollusk effectively either piercing, cutting or sawing the algal cell wall depending on its thickness and absence or presence of trabecular reinforcements (Fig. 1, bottom).Adaptive dietary radiation among sacoglossan families shows a correlation between radular teeth 228 A. MARÍN and J.D. ROS Elysia timida y E. viridis, al igual que las especies de Limapontoideos, se caracterizan por su capacidad de sintetizar de novo metabolitos tales como polipropionatos.Un análisis detallado de la cleptoplastia y las defensas químicas en los Sacoglosos puede proporcionar una mejor comprensión de la ecología y evolución de estos opistobranquios especializados.En este artículo se resumen algunos de los últimos descubrimientos, relacionados principalmente con especies mediterráneas, y se ofrece una hipótesis evolutiva plausible sobre la base de las tendencias biológicas y químicas que podemos distinguir en ellas.
Palabras clave: Sacoglosos, defensa química, evolución.length and algal cell diameter (Fig. 2).After puncturing the alga, they extract the cell-sap with a muscular pharyngeal pump (Jensen, 1991).The digestive system is adapted to a fluid diet, with a large internal surface area for absorption and a reduced intestine and anus.The digestive gland tends to be highly branched, particularly in those sacoglossans that retain functional chloroplasts from their host plants.
Sacoglossans display a remarkable diversity in shape, ranging from shelled burrowing species (Ascobulla) to epifaunal shell-less forms such as Elysia, Ercolania, and Caliphylla.The Sacoglossa are divided into two major classes, shelled sacoglossans (Suborder Oxynoacea) and shell-less sacoglossans (Suborder Plakobranchacea; Williams and Walker, 1999).The taxonomic classification used here is based on the phylogenetic analysis made by Jensen (1997a;Table 1).
In this work we review the factors selecting for host specialisation and the role of secondary metabolites in the evolution of these specialist herbivores, with an emphasis on the chemical ecology of Mediterranean sacoglossans.

ORIGIN OF SECONDARY METABOLITES IN SACOGLOSSANS
Most shelled mollusks are protected from predation by a hard shell and do not possess any special secondary metabolites that might function as a chemical defense.However, most opisthobranchs lack the physical protection of a shell yet they compensate for their physical vulnerability with chemical secretions, nematocyst-based defenses or a cryptic coloration (Ros, 1976;Todd, 1981, etc.).The primitive shelled Sacoglossans have both a shell and the ability to produce a chemical exudation.The main source of the chemical defense in opisthobranch mollusks resides in the selective concentration of chemicals contained in their food.The presence of molecules structurally related to typical dietary metabolites in Sacoglossans that feed on algal species from the order Caulerpales could be attributed to the selective accumulation of minor compounds, or to an in vivo chemical transformation of major metabolites, both acquired from the diet.The Limapontioidea and some Plakobranchidae species are characterised by their ability to synthesize polypropionate metabolites.Secondary metabolites generated via the propionate route are quite unusual in nature but rather common in marine mollusks, where they have been found in Cephalaspi-  deans, Anaspideans, Notaspideans, Pulmonates and Sacoglossans (Faulkner, 1992;Cimino and Sodano, 1993).When disturbed, the elysioids Elysia timida and E. viridis produce a defensive mucus that contains polypropionate metabolites (Gavagnin et al., 1994b(Gavagnin et al., , 1994c)).These species are known to feed on algae of the genera Acetabularia and Codium, which, however, do not contain any polypropionates.Lack of evidence for a food-linked origin of these metabolites lends weight to the hypothesis that Sacoglossans have developed the ability to biosynthesize their defense allomones.

SECONDARY METABOLITES OF CAULER-PALEAN ALGAE
Many species of caulerpalean algae grazed by sacoglossans contain secondary metabolites which show biological activity towards microorganisms, invertebrate eggs, sperm, larvae, fishes and mollusks (Paul and Fenical, 1986).Most of the compounds discovered in the algal families Caulerpaceae and Udoteaceae are sesquiterpenoids and diterpenoids, and are often acyclic.Enol acetates, aldehydes and the terminal bis-enol acetate moiety are common to most of these metabolites, which are functional groups with high biological activity and chemical reactivity.These structural features may be responsible for the wide spectrum of biological activity these metabolites show toward microorganisms, invertebrate eggs, sperm, and larvae, marine fish, and mollusks.Thus, the ancestral sacoglossan would have been able to colonize an algal substratum full of active secondary metabolites which acted as deterrent against generalist herbivores, but not to specialist ones like this early sacoglossan.The first adaptation of the primitive sacoglossan that allowed it to exploit Caulerpa might have been to develop a mechanism for the detoxification of the biologically active sesquiterpenoids and diterpenoids.In this way, the ancestral sacoglossan would have gained a free-space without competitors (meso-and macroherbivores) and potential predators.
Several sesquiterpenoids and diterpenoids have been isolated from the ancestral food of Sacoglossans, the pantropical and subtropical genus Caulerpa (Fig. 3).The Mediterranean Caulerpa prolifera contains the acetylenic sesquiterpenoid caulerpenyne, which is toxic and a feeding deterrent against generalist herbivores such as mollusks or sea urchins (Amico et al., 1978;Mc Connell et al., 1982;Paul and Fenical, 1986), although it does not protect the alga against tropical herbivorous fish (Meyer and Paul, 1992).Caulerpenyne has also been found in other common tropical Caulerpa species, in both the Caribbean Sea (C. racemosa, C. mexicana, C. sertularioides, C. paspaloides, C. lanuginosa, C. cupressoides, and C. verticillata) and the Pacific Ocean (C.racemosa, C. taxifolia, and C. cupressoides).Flexilin is a sesquiterpenoid closely related to caulerpenyne, isolated from the southern Australian species Caulerpa flexilis.Both caulepenyne and flexilin possess the 1,4-diacetoxybuta-1,3-diene (bis-enol acetate) moiety.This functional group is unknown in terrestrial species, but it is common in the majority of green algal metabolites.Although some of the first chemical studies on the genus Caulerpa described caulerpicin and caulerpin, their exact biological role has been disputed by several authors.Caulerpin is a bright yellow-orange compound, which has been found in more than half of the investigated Caulerpa species (Vest et al., 1983).Although this molecule was originally described as a bioactive metabolite, recent studies suggest that it lacks functionality.Conflicting chemical evidences indicates that caulerpicin does not represent a single pure compound.
Surprisingly, many of the metabolites isolated from the family Udoteaceae have been found to be closely related to, or identical with, those described from Caulerpa species (Fig. 3).The sesquiterpenoid rhipocephalin was isolated from the common Caribbean alga Rhipocephalus phoenix.Rhipocephalin is closely related to the Caulerpa prolifera metabolite, caulerpenyne, but lacks its acetylenic functionality.The sesquiterpenoid flexilin from C. flexilis has also been reported in the Caribbean alga Udotea conglutinata and in the Pacific species U. geppii.Studies of the abundant Mediterranean alga Udotea petiolata led to the isolation of udoteal, which has been described in U. flabellum from the Caribbean Sea, and U. argentea from the Western Pacific.Petiodial has also been reported in U. petiolata from the Mediterranean Sea.
Most species from the genus Halimeda contain halimedatrial, a cyclopropane-containing trialdehyde (Paul and Fenical, 1984).The major metabolite isolated from the Mediterranean alga Halimeda tuna is halimedatrial.The Mediterranean alga Derbesia tenuissima produces an epoxylactone previously isolated from the cosmopolitan species Pseudochlorodesmis furcellata.This compound is structurally related to the epoxylactone produced by Udotea argentea.
It is not clear what chemical features determine the degree of deterrence or toxicity of caulerpalean metabolites.Compounds that differ only slightly in chemical structure can vary greatly in their deterrent effects (Hay et al., 1988).The closely related green algal metabolites udoteal, flexilin, chlorodesmin and caulerpenyne differ in their feeding deterrent effects even though all contain the same major bis-enol acetate functional group (Paul et al., 1988b).

CHEMICAL DEFENSE IN SHELLED SACOGLOSSANS
Shelled sacoglossans (about 50 species, belonging to four families) feed on algae of the genus Caulerpa (Clark et al., 1990;Gavagnin et al., 1994a).This specialized diet of Caulerpa, the presence of a shell, non-functional chloroplast retention, a chemical defense derived from the diet and burrowing habits, could be considered primitive characters within the Sacoglossa.The most primitive sacoglossan genus, Ascobulla, and some species of Volvatella, live below the sediment surface.The remaining shelled species are all epialgal but also feed on Caulerpa.These primitive members accumulate chemicals from their diet.The transfer of toxic metabolites from caulerpalean algae to grazer sacoglossans was recognized in 1970 by Doty and Aguilar-Santos in Oxynoe panamensis, which accumulates caulerpicin and caulerpin from Caulerpa sertularioides.
In the Mediterranean Sea, the endemic sacoglossans Oxynoe olivacea, Lobiger serradifalci and Ascobulla (=Cylindrobulla) fragilis feed selectively on different parts of the alga Caulerpa prolifera (Gavagnin et al., 1994a).Many species of sacoglossans that feed on caulerpalean algae have shapes that closely match those of the algal food.Oxynoe olivacea and Lobiger serradifalci display homochromy, whereby the pigments contained in the mantle give them an identical colour to that of the alga Caulerpa prolifera.The infaunal A. fragilis lives below the sediment surface where it feeds upon the runners of C. prolifera.This primitive sacoglossan maintains the mechanical defensive role of the shell fully functional, and when the animal is dis-turbed its body can be retracted completely into the cylindrical shell.However, when alarmed the mollusk ejects an opalescent white defensive fluid.When disturbed, species of the genus Lobiger cast off their parapodia in a process known as autotomy, while species of the genus Oxynoe shed their tail since their shell has become reduced in size, allowing the head to be retracted but not the tail or parapodia.The host plant Caulerpa prolifera contains the sesquiterpenoid caulerpenyne as the main component.O. olivacea and A. fragilis are able to modify caulerpenyne into more toxic compounds, oxytoxin-1 and oxytoxin-2, characterized by the pres-ence of aldehyde groups (Cimino et al., 1990).Caulerpenyne is converted into oxytoxin-1 and oxytoxin-2, which are located in the mucus and external tegument of A. fragilis and O. olivacea.However, Ascobulla fragilis retains unmodified caulerpenyne in the digestive gland as a primitive character.L. serradifalci only contained oxytoxin-1 in the parapodial lobes and in the defensive mucus but neither caulerpenyne nor oxytoxin-2 were found.Feeding assays with caulerpenyne demonstrated that this metabolite did not act as a deterrent towards the marine fishes Thalassoma pavo, Chromis chromis and Serranellus hepatus (Gavagnin et al., 1994a(Gavagnin et al., , 1994b)).These three caulerpivorous sacoglossans are thus able to modify the main algal metabolite into the more toxic metabolites oxytoxin-1 and -2 (Fig. 4).The defensive allomone oxytoxin-1 has been found to be toxic and to act as a feeding deterrent to natural fish predators.Due to the high chemical reactivity of oxytoxin-2, it was not possible to test the pure compound in identical conditions to caulerpenyne and oxytoxin-1, but the feeding assays realized directly with the mucus secreted by O. olivacea, containing a mixture of oxytoxin-1 and -2, resulted highly deterrent to Mediterranean carnivorous fish.

THE CHEMICAL DEFENSE OF PLAKOBRANCHOIDEA
The Plakobranchoidea (=Elysioidea) consist of parapodium-bearing taxa.The species belonging to the genus Elysia have a narrow foot and a pair of lateral, leaf-like parapodia, often green in color, in which the sequestered algal chloroplasts are retained.Many species are generally found in association with green algae but some species include other algae and even seagrasses in their diet (  udoteal and halimedatrial tetraacetate derived without a chemical biotransformation from the algae they eat (Gavagnin et al., 1994b).These diterpenoids are characterized by a protected 1,4 conjugated dialdehyde system and they exhibit cytotoxic, antimicrobial and feeding-deterrent activity (Paul and Fenical, 1987).The sacoglossan Elysia translucens feeds on the caulerpal Udotea petiolata, and kleptoplasty confers on it the same green colour.
Comparative chemical analysis of Elysia translucens and the host plant U. petiolata shows that the sacoglossan accumulates large quantities of udoteal (Paul et al., 1982), the main metabolite of the alga, in defensive glands of the parapodia.The diterpenodid halimedatrial tetraacetate is produced by the caulerpalean Halimeda tuna.Bosellia mimetica feeds on H. tuna, and its flat shape and green parapodia (due to chloroplast retention) camouflage it well on the alga.TLC analysis of Bosellia mimetica and of its prey H. tuna pointed to strong similarity between the two metabolic patterns, the major secondary metabolite of the former being halimedatrial tetraacetate, which was previously identified in the host alga H. tuna.Many sacoglossans are not cryptic on their algal food or usual substrate and make no attempt to conceal themselves, which may suggest that they show aposematic or warning coloration.However, there are no experimental justification for this and the most strikingly colored sacoglossans are cryptic due to disruptive coloration which breaks up the animal's shape.The Mediterranean plakobranchid Thuridilla hopei shows a brightly colored pattern in the laboratory with a dark green or black mantle crossed with blue, red and yellow lines.T. hopei is an endemic species from the Mediterranean Sea, which is found in association with the caulerpal Derbesia tenuissima, whose principal secondary metabolite is a diterpenoid also isolated from the cosmopolitan alga Pseudochlorodesmis furcellata (Paul et al., 1988a).The same algal metabolite was found in T. hopei, together with other three related diterpenoids, thuridillin-A, -B, and -C (Gavagnin et al., 1993).The chemical analogy of the three thuridillins with the primary algal metabolite suggests that the sacoglossan feeds on D. tenuissima and modifies its dietary metabolites with the aim either of reducing their toxicity or of transforming them into more active compounds (Fig. 5).
Polypropionates are rare in nature, but rather common in marine mollusks, where they have been found in Cephalaspideans, Pulmonates and Sacoglossans.Studies of the sacoglossan species Elysia (=Tridachiella) diomedea and Elysia (=Tridachia) crispata from the Caribbean Sea resulted in the identification of a novel group of propionatederived γ-pyrones (Ksebate and Schmitz, 1985;Ireland and Faulkner, 1981;Faulkner, 1992).The presence of polypropionates has also been described in the genus Elysia.Biosynthetic experiments with labelled sodium [1-14 C] propionate confirm that the allomones of the Mediterranean species Elysia viridis and E. timida are biosynthesized de novo by the mollusks.The ether extract of E. viridis was found to contain the polypropionate elysione, previously reported in the literature (Dawe and Wright, 1986).This polypropionate is the main component of the defensive mucus secretion of E. viridis.The comparative analysis of the algal food Codium vermilara and Elysia viridis demonstrated that the polypropionates have a de novo origin.In the biosynthetic experiments carried out with E. viridis, sodium [1-14 C] propionate was injected into the mollusks.After 24-72 h, the 14 C activity increased in the fractions containing elysione (Fig. 5).

DEFENSE MECHANISMS IN LIMAPONTIOIDEA
The superfamily Limapontioidea (=Polybranchioidea; =Stiligeroidea) contains the cerata-bearing sacoglossans.A comparative study of Mediterranean Limapontioidea revealed a correlation between the occurrence of defensive mechanisms and the morphoanatomical or biochemical features of the cerata of these species (Table 2).Defensive autotomy is a defense strategy largely used by opisthobranch mollusks as the last resource against predation (Stasek, 1967;Ros, 1976).This behaviour has adaptive significance in the prey organism when the autotomized structure: (a) is not essential for the continued existence of the prey itself; (b) is the most frequently attacked part of the prey, and (c) preferably contains the most powerful deterrent substance (Todd, 1981).Thus, detachment of the tail, or parapodia, in the shelled genera Oxynoe and Lobiger, or the cerata in some species of Limapontioidea, can be very effective in deterring predators, especially if accompanied by the emission of large amounts of toxic mucus from the autotomized appendages.
The Mediterranean species Cyerce cristallina is a conspicuous sacoglossan that resembles an eolid opisthobranch because its slender body is flanked by voluminous cerata.Each ceras is flattened and observed to be covered by red bands on a white background.When the animal is attacked by predators, the cerata are detached from the mantle and exhibit prolonged contractions while secreting large amounts of toxic mucus (Di Marzo et al., 1991;Vardaro et al., 1991).After the autotomy, the cerata are regenerated within 7-10 days.The internal structure of the cerata exhibit peculiar adaptations to defensive autotomy.Electron microscopy has shown that the flattened cerata do no contain branches of the digestive gland as is the case for other cerata-bearing sacoglossans and, therefore, the origin of the white and red colour of the appendages cannot be attributed to rhodoplast retention.The major part of the volume of the appendages is filled by cells with an enormous central vacuole, with no haemocoelic spaces.The cerata contain both transverse and longitudinal muscles, which are responsible for its prolonged contractions, and sphincter-like muscle fibres located at the base of each ceras, which cause the appendage autotomy.The body of the limapontiid Ercolania funerea is translucent, and has several latero-dorsal rows of cerata.Each ceras is rounded and white spotted, with green digestive gland branches appearing through the skin.The ceratal green colour is due to the presence of intact chloroplasts in the digestive cells sequestered from the chlorophycean Chaetomorpha linum.Among the ciliated cells, the epithelium contains unicellular glands, which are probably responsible for the secretion of slime.Both longitudinal and transversal muscular fibers, inserted in broad haemocoelic spaces, are also found between the epithelium and the digestive gland, and, as in Cyerce cristallina, this musculature is better developed at the base of each ceras, where it forms sphincter-like structures which, when the mollusk is disturbed, contract strongly, thus cutting off the cerata.
In Placida dendritica both the albumen gland and the digestive gland protrude into the cerata.The ceratal digestive gland branches contain functional chloroplasts identical to those of the algal food Bryopsis plumosa.The cerata are covered with a ciliated epithelium which is intercalated with unicellular defensive glands.The base of the cerata is solid with no special muscular system.The cerata of Calliphylla mediterranea are leaf-shaped and transparent, so that the green branches of the digestive gland can be discerned.These branches show intact chloroplasts sequestered from the algal food, B. plumosa.The albumen gland does not protrude into the cerata.Other histological features include the presence of unicellular glands that are not located in the proximity of the epithelium and the absence of sphincter-like muscles at the base of the cerata.When molested, C. cristallina and E. funerea produce a defensive mucous secretion and can easily shed their appendages.However, P. dendritica and C. mediterranea normally do not autotomize the cerata if they are disturbed.For a comparative analysis of the rate of regeneration among species, the cerata were removed by means of dissecting forceps from specimens of P. dendritica and C. mediterranea.After loss of the cerata, spontaneous or aided with dissecting forceps, the fastest regeneration was observed in C. cristallina and E. funerea (8 to 10 days), and the slowest in P. dendritica (15 days).C. mediterranea showed an incipient regeneration, but it was too slow to be measured (> 30 days).Histological sections of regenerated cerata of C. cristallina and E. funerea exhibited the same appendicular structure and the same cell types as the fully developed cerata.
C. cristallina, E. funerea and P. dendritica produce αand γ-pyrone polypropionates while C. mediterranea does not contain polypropionates (Vardaro et al., 1991(Vardaro et al., , 1992a(Vardaro et al., , 1992b;;Di Marzo et al., 1991).These secondary metabolites have structures that differ only in the degree of methylation and the geometry of the double bonds of the side chain and are only distributed in the cerata and defensive slime of these species (Fig. 6).Experiments to evaluate the role of polypropionates in cerata regeneration indicated that the most active compounds were the ones with an all-trans-alkyl chain, that were found specifically in the cerata of the quickly regenerating species (C.cristallina and E. funerea), while the γ-pyrones with inverted geometry of double bonds isolated from P. dendritica were either not active or very weakly active in cerata regeneration.The defensive mucus produced by C. cristallina, E. funerea and P. dendritica were toxic to the fish Gambusia affinis, while the slime of C. mediterranea was harmless.Therefore, polypropionate metabolites from C. cristallina and E. funerea may function to prevent predation and as inductors of cerata regeneration.
The defensive mechanisms of these four Limapontioidea species show the evolutionary plasticity of Sacoglossans in exploiting different algal resources.Calyphylla mediterranea feeds on algae pertaining to the genus Bryopsis, all of which are coenocytic species with a morphology close to the primitive food Caulerpa.This sacoglossan retains functional chloroplasts but does not exhibit autotomy nor chemical deterrence.Apparently, chloroplast-provided camouflage is the only means of defense in this species.Cryptic behaviour is also used by Placida dendritica and Ercolania funerea.The ceratiform P. dendritica grazes on the coenocytic thallii of Bryopsis and Codium algae.The defense of this species is based on the exudation of large vol-umes of pyrone-containing slime, which generates a defensive "cloud" around the mollusks.The defensive secretion is accompanied by defensive behavior when the animal is molested, and the cerata are erected and waved frantically.The small sacoglossan Ercolania funerea retains non-functional chloroplasts from the cladophoral Chaetomorpha linum.E. funerea combines several defensive strategies found in Limapontioidea species: cryptic coloration, autotomy, ceratal regeneration and chemical deterrence (Vardaro et al., 1992a).The cerata-bearing Cyerce cristallina exhibits the most evolved defensive strategy.The autotomy of the cerata, which in this species do not contain any prolongation of inner organs and can be compared to large reservoirs containing toxic polypropionates, has been highly improved.C. cristallina detaches its cerata when attacked by predators; the detached cerata exudate a large volume of deterrent mucus and contract visibly, thus diverting the predator from the animal body to the contracting cerata.

EVOLUTION OF KLEPTOCHEMISTRY AND KLEPTOPLASTY IN SACOGLOSSANS
The evolution of Sacoglossans has lead to trophic specialisation, which is a characteristic of species of mature ecosystems with high species diversity.The species diversity of the Mediterranean Sea is impoverished relative to the Caribbean Sea or to the tropical Indo-Pacific Ocean.There are fossil evidences that Sacoglossans could be one of the "oldest European habitants".The first record of a sacoglossan fossil belongs to the "bivalve" Berthelinia (Juliidae) from the Eocene of the Paris Basin (Keen and Smith, 1961).Valdés and Lozouet (2000) have described the fossil record of four shelled species in a exceptionally well-preserved collection of Tertiary from the Aquitaine Basin, France.The Tertiary European sacoglossans include three species of the family Juliidae, Julia girondica, Squamulina sp. and Berthelinia sp., and one of the family Volvatellidae, Volvatella faviae.The presence of these fossils indicate the probable existence of algae of the genus Caulerpa in this shallow sea due to the close trophic relationship between shelled sacoglossans and this genus.Most species of Caulerpa are uncalcified and do not leave direct remains of their presence.Definitive separation of the Indo-Pacific and Atlantic species occurred during the closure of the main East-West connection, during the Oligocene-Miocene transition.The geographical range of the genus Volvatella is today restricted to the tropical Indo-Pacific and Caribbean, being absent from Europe and West Africa (Valdés and Lozouet, 2000).This discontinuous geographical range is all that remain of a wider distribution through the Tethys Sea.The extinction of this high diversity of shelled sacoglossan species in Europe might have occurred during the first major cooling period in the mid Miocene (Lozouet et al., 1994).From this point of view, there is a general tendency to think of the Mediterranean sacoglossans only as a relictual fauna or even as a group of invasive species from tropical areas.However, we believe the Mediterranean sacoglossans are a particularly instructive example of how trophic specialisation in ancestral sacoglossans from the Tethys Sea have lead to similar defensive and chloroplast exploitation strategies in different geographical areas.
For Sacoglossans, one may think of the following evolutionary history.First of all, we must place ourselves in the paleo-scenario of the tropical sea that was the Tethys.In this time, the shallow, well light and warm waters favored the growth of uncalcified algal meadows, made up from caulerpacean species chemically defended against generalist herbivores.The ancestral sacoglossan must have developed a diet-derived chemical defense mechanism prior to the shrinking or loss of the shell.However, it must first have overcome the secondary metabolites contained on the ancestral food, algae of the genus Caulerpa, to deter generalist herbivores.The shelled sacoglossans developed a mechanism to sort sesquiterpenoid and diterpenoid metabolites from Caulerpa into those that could be catabolized and those that were noxious or toxic.The sesquiterpenoids or diterpenoids were then transferred to defensive glands of the mantle, from which they could be excreted in response to an attack.The burrowing species of Volvatellidae, including the genera Volvatella and Ascobulla, could be considered an evolutive link between the infaunal cephalaspideans and the most advanced sacoglossans (Clark et al., 1990).Only when the mechanism of chemical exudation in defensive glands was fully operative could the ancestral burrowing sacoglossan exploit the aboveground, erect parts of Caulerpa.Oxynoe olivacea and Lobiger serradifalci living and thriving on Caulerpa prolifera are modern examples.Jensen (1997b) suggests that the original host could be other than Caulerpa because she found Cylindrobulla phuketi feeding upon algae of the genus Halimeda.However, if we compare the parallel evolution in the Indo-Pacific, Atlantic and Mediterranean areas, the initial association with Caulerpa seems to be the more probable starting point.We do not found arguments enough to change the traditional scenario for this alternative hypothesis.Fossils of the udoteacean alga Halimeda date back to the Cretaceous (Hillis-Colinvaux, 1980).Fossils of calcareous algae of the order Dasycladales are usually associated to the Tethyan transgression.The possible paleoecological scenario having changed, the majority of uncalcified caulerpaceans became extinct due to interspecific competition, feeding by herbivores or climatic changes.The present-day tropical seaweeds on coral reefs are heavily grazed by generalist herbivorous species (fish, sea urchins and gastropods), which thus can seize from 60 to 100 % of total production (Hay and Steinberg, 1992).Several studies have shown that increasing the mineral content of food plants by adding powdered calcite and/or aragonite can strongly deter feeding by some herbivores.In Halimeda, the CaCO 3 crystals in the thallii fuse to form a larger skeleton which has an additional negative effect on feeding by herbivorous fishes and sea urchins (Pennings and Svedberg, 1993).Many actual calcified caulerpacean algae are also chemically defended.The selection should favour seaweeds with structural and/or chemical defenses active against numerous types of generalist herbivores.The evolutionary adjustment to generalist herbivores probably exerted a parallel selective pressure for the evolution of sacoglossans.Here the metaphor of the Red Queen to describe biotic evolution (Van Valen, 1973) is of application: the consistent change is necessary, not to increase adaptiveness, but merely to maintain it.
The evolutionary trend observed in Mediterranean Plakobranchoidea suggests that the next event could have been parapodium-bearing species with kleptoplasty colonizing other caulerpalean algal species with structurally similar chemicals and robust chloroplasts.The change of the shelled sacoglossans from a Caulerpa-based diet to other green algae may have involved a very small genetic change in Plakobranchoidea species.Chloroplast retention in the ancestral epialgal sacoglossans probably functioned as nutritional homochromy, in which intact chloroplasts provided a cryptic coloration identical to that of the host alga.There is a convergent evolution between functional plastid retention and the origin of secondary metabolites of Sacoglossans.Siphonalean green algae are known to contain secondary metabolites, which act as feeding deterrents against generalist herbivores (Norris and Fenical, 1982).Most "primitive" Mediterranean Plakobranchoidea, Elysia translucens and Bosellia mimetica, accumulate udoteal and halimedatrial from the caulerpalean species Udotea petiolata and Halimeda tuna respectively, which are metabolites closely related to those described from Caulerpa species.E. translucens and B. mimetica retain intact chloroplasts which provide a coloration virtually identical to the algal background.In this pattern, more advanced taxa feed on other Siphonales, Siphonocladales, Cladophorales and other exotic foods.With the dietary radiation of the Sacoglossans, the chemical defense mechanisms probably adjusted to the habitat characteristics.Some species maintained the ability of kleptoplasty with a chemical defense derived from the diet.The elysioid Thuridilla hopei probably modifies algal metabolites to produce a more effective antifeedant chemical and retains functional chloroplasts.The exploita-tion of algal plastids in shell-less sacoglossans seems associated with the amount of cell sap that the host plant provides to the slug due to the own algal arquitecture (e.g., uniseriate filamentous, blade fronds), to structural defenses (e.g., calcification), or low host plant density (Fig. 7).An example of the evolutionary pressure that food limitation can exert on sacoglossans to acquire functional chloroplasts is to be found in the relationship between the elysioid Elysia timida and the unicellular alga Acetabularia acetabulum (Marín and Ros, 1992).Elysia timida is an stenophagous herbivore that feeds exclusively on the green alga Acetabularia acetabulum.In the Mediterranean Spanish coast, the life cycle of the alga begins in October, when the young singlecelled plant has two growing apices, one giving rise to the rhizoidal system that attaches the plant to the substrate, the other growing apex becoming the erect thallus.Starting in January, the cell walls of the thallus undergo progressive calcification; by April the base of the thallus is very highly calcified, although the apex continues its vegetative growth.The apex does not calcify until June, when the thallus vegetative growth stops and a cap of gametangial radial chambers is formed.The gametangial rays contain numerous cysts which when mature release pyriform biflagellate isogametes.The calcification process is not simultaneous; algae whose thallii are calcified to different extents coexist in the same population at any one time, and different A. acetabulum populations also feature an unequal calcification period.Elysia timida features a whole series of adaptations for optimum kleptoplast exploitation.Its photosynthetic production is one of the highest amongst the Mediterranean sacoglossans and in the laboratory it can survive for up to three months when starved.All these adaptations are related to the seasonal dynamics of the food used.The young E. timida can be found on shallow rocks when the first A. acetabulum stalks appear in October.The population density of the sacoglossan species then increases to a maximum in December and a secondary maximum in May; the mollusks disappear in summer.The combination of number of individuals and individual growth produces a maximum sacoglossan biomass out of step with the population maximum.Calcification of the alga cell wall first complicates browsing by E. timida, then impedes it altogether.Between January and June, the sacoglossan moves to the non-calcified apices of the algae, where it eats.From June, when all the tallii are totally calcified, the E. timida population falls drastically, until it disappears completely the following month.Progressive calcification of the alga not only diminishes the food supply, but also makes it more difficult to find.If we divide the total number of stalks of A. acetabulum found in a given month by the number of non-calcified stalks, we obtain a parameter measuring the energetic effort the mollusks must devote to obtain the same amount of food.Even in March, virtually all attempts to find and eat a non-calcified stalk would have a positive result.In April, however, to obtain the same amount of food E. timida would have to double efforts, and in May the effort necessary to obtain the same amount of food would be nearly ten times greater than in autumn and winter.The effort would become nite in summer, when all Acetabularia stalks are calcified.This may explain the disappearance of the slug by mid-summer.
The food resource limitation hypothesis exposed previously to try explain the role of kleptoplasty in Sacoglossans must be complemented, however, with the defensive role which the cryptic coloration that sequestered algal plastids confer to these mesoherbivores.Aposematic coloration patterns have been only described in sacoglossans that feed on seaweeds with patchy distribution or whose small size make useless a cryptic pattern on a variable background.Ghiselin (1966) and Jensen (1996) reject the view of some authors that the shell-less sacoglossans are polyphyletically derived from shelled forms in favour of treating the shell-less forms as a single clade.This hypothesis suggests that Limapontioidea and Plakobranchoidea had a common shell-less ancestor, in which polypropionate metabolites together with chloroplast retention first appeared.But these characters could have derived separately from the common ancestor with a convergent evolution of polypropionate biosynthesis and functional kleptoplasty in each line.The Red Queen effect referred to above lead us to consider that the de novo biosynthesis of polypropionates by Sacoglossans is forced by the host-plant specialisation of these mollusks where kleptochemistry is not possible, and is the result of their selective predation.
FIG.3.-Types of secondary metabolites found on coenocytic green algae consumed by Sacoglossans.
FIG. 5. -Origin of secondary metabolites in Plakobranchidae (=Elysiidae): A, biotransformation of active compounds sequestered from the food in defensive allomones; B, selective accumulation of algal metabolites; C, biosynthesis de novo of their own chemical defenses.

TABLE 2 .
-Algal diet, kleptoplasty, defensive behaviour, larval development and secondary metabolites with their biological role in Mediterranean sacoglossans.