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4. Effects on autochthonous species and C.taxifolia's toxicity

As C.taxifolia colonizes all types of substrata from the eulittoral down to 100m depth, inducing a homogenization of microhabitats and a reduction of the architectural complexity of the substratum (4.1Harmelin, 1996; 4.2Harmelin-Vivien et al., 1999), a decrease in diversity and abundance of motile invertebrates was observed in the C.taxifolia meadows (4.3Bellan-Santini et al., 1996), as well as a persistent decrease in mean species richness, density and biomass of fish assemblages (4.4Francour et al., 1995; 4.2Harmelin-Vivien et al., 1999), when compared to indigenous communities living in P.oceanica beds or in rocky areas. Relini et al., 4.5(2000) described qualitative and quantitative changes in fish communities during the replacement of the sea grass Cymodocea nodosa with that of C.taxifolia (at Imperia, western Ligurian Sea, Italy). In addition to the high fishing pressure (local/coastal fishing industry) and weak rugosity, they strongly impacted upon density and demographic structure of fish assemblages and ultimately leading also to a decrease of the abundance of larger-sized fishes.
As will be outlined in this part, the possible transfer of toxins through the food chain presents a toxicological risk not only for marine organisms exposed to it but also for man; i.e. certain mollusks feeding on Caulerpa showed a two- to three-fold concentration of toxic metabolites and became themselves toxic to predators, while human food poisoning resulting from the consumption of the Mediterranean bream Sarpa salpa has been already observed (4.6Spanier et al., 1989, 4.7aWhitehead et al., 1986).

4.a. Effect on microbial loop: Soft sediment bottoms are not only characterized by a high infauna and epibenthic fauna, but are directly coupled to the microbial association of the sandy substrate. Particulate organic material and plankton organisms in the surface water are trapped and accumulate temporarily in shallow soft bottom sediment. Usually some of this carbon is available for consumption by benthic microbes in the system. However, the presence of an algal mat on otherwise unvegetated shallow soft bottoms considerably changes its ecosystem structure and functions. Rather than enabling a healthy microbial association to perform the nutrient conversion and especially under eutrophicated conditions, C.taxifolia takes over the "recycling activity". The altered nutrient dynamics of the sediment becomes evident in the net accumulation of organic matter. In the long term however, the organic enrichment leads to higher oxygen consumption that, together with the reduced water exchange, will result in decreased oxygen levels in both the water column and the sediment. The reduced oxygenation of the sediment causes the redoxcline to move towards the sediment surface further reducing the mineralization-capabilities of the microbial loop (4.7bTroell et al., 2004).

4.b. Effect on flora: Phytobenthic studies of highly infested areas around Cap Martin (Southern France) showed a drastic impoverishment of autochthonous communities. The creeping and erect axes of C.taxifolia shade off the light while its rhizoids trap and chemically alter the sediment. Most autochthonous algae tend to disappear quickly while crustose algae seem to be eliminated latest. The paucispecific C.taxifolia meadows tend to substitute for all sheltered algal infralittoral phytocoenosis which stands for a dramatic fall of richness and diversity of the littoral ecosystem.
Among the dozens of plants that are found in an intact Mediterranean ecosystem are the marine phanerogams of Posidonia, Cymodocea or Zostera.
Posidonia oceanica is a dark, gray/green endemic sea grass covering large areas of the seabed at depths between 30-40m (4.8Boudouresque & Meinesz, 1982), thus a fundamental key sea grass for that ecosystem. Posidonia meadows, like the other species of sea grass, not only bolster and protect the coastline, but it is one of the most important coastal primary producers (4.9Ott, 1980), act as a refuge, habitat, substrate for epiphytes, providing food and shelter for a huge variety of fish and invertebrates, and is the spawning ground and nursery for a countless number of species (4.10Meinesz, 2000 - see epiphyteic growth, fig.4).
Although P.oceanica possesses phenolic compounds that play an important role in the protection of these plants against competitors, predators and pathogens, that increase in stress-situations (4.11Agostini et al., 1998), it does not seem to effectively deter C.taxifolia from invading areas dominated by the seagrass P.oceanica; thereby killing up to 45% of Posidonia shoots in one year (4.12Villele & Verlaque, 1994). Similarly, other native seagrass (e.g. Cymodocea nodosa) are being more or less totally replaced (4.5Relini et al., 2000; 4.13Ceccherelli & Cinelli, 1997).
Since the phases of vegetative growth in P.oceanica and C.taxifolia are reversed (4.14Boudouresque et al., 1995; 4.15DeVillele and Verlaque, 1995) the leaves of P.oceanica are larger in winter and spring than the fronds of C.taxifolia, which corresponds to the period during which growth of the latter species is at a minimum (4.16, 4.17Meinesz et al., 1993, 1995).


Fig.4 Epiphytic growth on P.oceanica 4.17aHofrichter, 2001; (180kB)
 

 
Fig.4.a A running stolon of C.taxifolia (above) and C.taxifolia competing with P.oceanica (below) (135kB)

Both live and dead rhizomes of P.oceanica have in fact proved to be excellent substrate for the settling and development of this invasive chlorophyte (4.18Cuny et al., 1995). Consequently the patchy leftovers of P.oceanica along the southern coast of France could not cope with these altered unfavorable abiotic conditions and regressed giving way to this invasive alga in shallow waters and leaving behind decaying plant tissues. Indeed, the scale of destruction of the Posidonia meadows, which are irreversible, is alarming. Based on those suggestion Cecherelli & Cinelli, (4.191999) predict that patches of P.oceanica at 10m depths are more vulnerable to algal invasions than at 2m depths. As P.oceanica leaves are moved by wave surge, they may sweep C.taxifolia from the substratum or abrade and tear it. If the negative effect of the whiplash is proportional to the length of seagrass leaves, it could explain the interaction of seagrass canopy height and density.

During the Californian infestation focus on diversity of organisms in the three vegetation types was not the main aspect of the study, yet Tippets (4.202002) was able to observe a decline in the richness between the native Californian eel grass Zostera marina and Caulerpa samples. A decline in certain "keystone" species, including amphipods, eventually results in a decline in all species dependent upon these organisms for food with ramifications extending up the food chain. As will be discussed later-on, one should not ignore the obvious presence in epiphytic end epizootic organisms commonly found on autochthonous seagrass species and as a result of the toxic cocktail of the allochtonous invader, the complete absence of such flora and fauna on its fronds. The effects of C.taxifolia invasions, and the ramifications on the benthic ecology of near shore lagoons, could be devastating to the basal levels of the food chain (4.20Tippets, 2002).


Fig.4.b Effects on autochthonous fauna (77kB)

Due to the out-competing properties of C.taxifolia (eutrophicated sediments, growth rate, gigantism, etc. see above, it is becoming the canopy forming species in the Mediterranean (4.17Meinesz et al., 1995; 4.22Ceccherelli and Cinelli, 1998); it occupies almost any substrate, and is therefore capable of replacing autochthonous floral competitors (4.23Verlaque and Fritayre, 1994; 4.24Ferrer et al., 1997).

4.c. Effect on fauna (invertebrates): Belsher & Meinesz (4.251995) observed that this invasive species suffocates numerous white Gorgonians Eunicella verrucosa at depths beyond 40m.
Bellan-Santini et al., (4.261996) analyzed C.taxifolia's effect on three important groups of invertebrates itself staple food for carnivorous fish: i.e. polychaetes, molluscs and amphipods are quantitatively and qualitatively dominant groups and at the same time exhibit varied types of trophic, reproductive, and dispersion strategies. Based on a low sampling effort, scientists have focused on results obtained by pooling the four seasonal samples at each location with the result that the numbers of individuals of mollusca, amphipoda and polychaeta in C.taxifolia meadows were greatly reduced. Aquaria observation revealed that after massive "bleeding" of a large Caulerpa-stand following spontaneous gametogenesis or mechanical injury, tubeworms behave abnormally. They will crawl half way out of their tube or even abandon the tube entirely, only to die shortly afterwards (4.35aDebelius & Baensch, 1997).

Pedrotti & Lemée (4.271999) could show that not only the marine microalga Cricosphaera elongata (phytoplankton) was subject to growth inhibition once incubated with organic extract of C.taxifolia. Even the herbivores feeding on it were affected. In particular a decreased survival rate of the sea urchin larva Paracentrotus lividus was found when the so exposed microalge was then offered to the larvae at different stages of development. Sensitivity to toxin-treated food depended on the larval stage at which exposure began. Larvae treated from first feeding (4-arm stage) with toxin-treated microalgae were most sensitive (25% survival, delay in development and metamorphosis of 32%). A C.taxifolia-diet begun at the 8-arm stage caused a decrease in survival and abnormal development; however, all the remaining larvae achieved metamorphosis (fig.4.c).
Boudouresque et al., (4.281996) could show the toxic effects on adult P.lividus. They showed that urchins feed during the summer / autumn experiment were affected, five urchins died during the 2nd month in the C.taxifolia containing aquaria. By the 3rd month, all the urchins showed sub-lethal signs, including immobility, very high righting times and large areas bare of spines. It is already known that C.taxifolia has a repulsive effect against various herbivores, and particularly against the tropical Atlantic urchin Lytechinus variegatus (4.29McConnel at al., 1982); however, series of experiments involving one of the most important macrograzer confirmed the negative impact of C.taxifolia upon herbivores - with the fish Sarpa salpa being placed at the macroscopic end of the herbivorous spectrum (4.30Amade & Lemée, 1998). Although this effect was stronger in the summer-autumn period, than during the rest of the year, most of the urchins even preferred to eat their own waste or resorted to pieces of plastic, rather than touching the algae. Such behavior caused the urchins to die of hunger rather than consuming the available C.taxifolia. These findings were and still are very disturbing. It simply means that algal endotoxins disrupt the entire food chain and biodiversity of the affected ecosystem.


Fig.4.c Cricosphaera elongata and P.lividus (75kB)

 


Fig.4.d Turnover rates of adult P.lividus (85kB)

4.d. Effect on fauna (fish communities): The objectives of some studies done so far were also done to quantify the consequences of this expansion on fish assemblages. It was feared that the bright color pattern of C.taxifolia might change predation pressure of carnivores feeding on herbi- and carnivorous fish communities; in fact fish color pattern is believed to serve three main functions: thermoregulation, communication and antipredator adaptation (4.31Endler, 1978; 4.31aCrook, 1997) and are the results of selection pressures on the genetic structure of populations. Color patterns represent a good tool in understanding the adaptive mechanisms of a species to its environment (4.31Endler, 1978, 1980; Planes & Doherty, 1997).

Relini et al (20004.30a), on the other hand, found that fish population changes in C.taxifolia meadows did not alter dramatically from those in P.oceanica meadows. Fish population still maintained the characteristics found in C.nodosa meadows and on sandy bottoms when C.taxifolia coverage was around 25%. Those authors concluded that the consequence of the allochthonous colonialization by C.taxifioia did not decrease fish biodiversity compared to autochthonous meadows. Bottom dwelling species (important commercial fish species) however suffered a severe blow due to the reduction of sandy seabed by the arrival of C.taxifolia.
In another study Francour et al., (4.332002) focused on the color patterns of individuals of four Mediterranean labrid species, Symphodus ocellatus, Symphodus roissali, Symphodus rostratus, and Coris julis, living in dense C.taxifolia meadows. They were compared with those of the same species inhabiting their usual indigenous habitats, the P.oceanica seagrass beds and the shallow rocky areas. In C.taxifolia meadows the proportion of green morphs observed in S.ocellatus and C.julis was significantly higher, particularly for small fishes. While S.ocellatus and C.julis settled in C.taxifolia meadows, S.roissali withdrew to shallow waters where C.taxifolia is not the dominant vegetation (4.34Francour et al., 1995).


Fig.4.e Changes in fish communities (450kB)

In its natural habitat, S.rostratus remained cryptic during the reproductive period, as it reproduced on the brown-dominated rocky area, whereas, in the new environment provided by the C.taxifolia meadows, reproductive individuals were more conspicuous to predators. Therefore, animals that rely on crypsis to avoid detection by predators commonly choose backgrounds upon which they will appear most cryptic (4.35Merilaita et al., 1999), by either moving away or trying to adapt via selection pressure - provided that they are able to survive on the altered food spectrum they rely upon.
An interesting observation requires to be mentioned: In modern public aquaria, Caulerpa-species are also kept and raised because of its "healing qualities", i.e. antibacterial effects. Sick fish will sometimes recover quickly in water that contains freshly squeezed Caulerpa-juice. This method is used in the modern Hasanal Aquarium in Brunei (4.35aDebelius & Baensch, 1997).

4.e. Toxic agents in C.taxifolia: C.taxifolia is native to the tropics thus subject of intense herbivore activity leading to the development of efficient chemical defense and antifouling capabilities (4.36Paul and Fenical, 1986; 4.37Faulkner, 1987; 4.38Schwede et al., 1987). The cocktail of repellent toxins consists of caulerpenyne (CYN), oxytoxins, taxifolials and other terpenes (Paul, 2002). As CYN is the most predominant toxin, it is believed that toxicity is almost exclusively based on the acetylenic sesquiterpene caulerpenyne (4.39Paul, 2002) with a bis-enol acetate functional group. Caulerpin, on the other hand, is a hydrophobic macromolecule containing a cyclo-octatetraene ring pigment (4.40Ayyad & Badria, 1994). Both chemical structures are shown in fig.4.f. This molecule is synthesized in the fronds of the algae, thus concentrations are higher in the erect blades than in the rhizoids, where they are released into the surrounding sea-water or consumed by herbivores (4.41Pesando et al., 1996). CYN exhibits antibiotic activity (4.36Paul and Fenical, 1986, 1987; 4.42Hodgson, 1984) and besides its cytotoxicity in mammalian cells including some eggs of some marine mammals (4.43Lemée et al., 1993; 4.44Pesando et al., 1994), it is toxic for molluscs, sea urchins, herbivorous fish, and capable of killing off many microscopic organisms and other epiphytic organisms. CYN extract inhibits or delays the proliferation of several phytoplanktons of the marine food chain (4.45Lemée et al., 1997).


Fig.4.f Structure of Caulerpenyne (CYN) and Caulerpin (40kB)

The aquaria strain of C.taxifolia contains higher CYN concentrations than the tropical strain (4.46Guerriero et al., 1992). C.racemosa as a Lessepsian migrant is likewise a tropical representative, but far less toxic than the C.taxifolia. The mean CYN values were found to be 80x higher in C.taxifolia than in C.racemosa (4.47Dumay et al., 2002) and are much higher than in other Caulerpa species (4.48Guerriero et al., 1994). CYN in the aquarium strain of C.taxifolia can account for up to 1.3% of the algal fresh weight or 2% or more of algal dry mass (4.39Paul, 2002).

4e. Toxicity of C.taxifolia: Under attack by herbivores - in the tropics by rabbit-fish (Siganidae) and surgeonfish (Acanthuridae; 4.39Paul, 2002) - the liberation of chemical precursors convert CYN into a potent feeding deterrent (4.49Jung & Pohnert, 2001). Since CYN is reportedly stable in pure seawater, the rapid conversion of it in an almost neutral environment (pH 6-7) suggests the involvement of an enzymatic reaction. This wound-activated enzymatic transformation enables C.taxifolia to store the less reactive CYN that can be transformed to aggressive metabolites when under attack. Since the reactive aldehydes are present within seconds after tissue damage, they might act locally as defensive metabolites during the relatively slow feeding process by slugs or sea urchins.

Possible metabolites after esterase-action from a wounded C.taxifolia on CYN are shown in fig.4.g (uppermost line); e.g. acidic deacetylation of caulerpenyne in the presence of 2,4-dinitro-ohenyl-hydrazine. For clarity tetraenes resulting from H2O elimination are omitted and only the most stable derivatisation product is shown (4.49Jung & Pohnert, 2001). Line 2, 3, and 4 of fig.4.g illustrate a few synthetic transformations from CYN isolated from C.taxifolia. However, no synthetic route toward CYN has been reported to date. Retrosynthesis scheme outlined the strategy for synthesizing CYN and one of its metabolites taxifolial A, which can be considered as a good precursor of CYN (4.50Parrain & Santelli, 2002).


Fig.4.g Caulerpenyne reactions (40kB)

Caulerpa's toxicity is highly seasonal; the heaviest disturbance occurred in summer and autumn when the size of C.taxifolia and its terpenoids production peaked at a maximum. Epiphytic and epizootic growth on C.taxifolia is insignificant except in spring when endotoxin concentration is lowest (4.51Amade & Lemée, 1998). The annual decrease in CYN concentrations, with the concomitant increase in frond length, under conditions of competition with P.oceanica is a likely result of a modified metabolism in the alga as the increased energy allocated to growth of fronds would be at the cost of another function (4.52Dumay et al., 2002).

Usually during late winter, the cover of C.taxifolia generally decreases (with algal fronds much smaller, 4.17Meinesz et al. 1995), only to pick up again during the warmer months to recuperate lost terrain. This periodic cycle correlates with its high growth rate, its total substrate occupation, light access, and sedimentation rates. This oscillation is reflected in the CYN concentration of the alga's frond wet weight: from to 0.2% in spring to 13% in summer (4.53Amade et al., 1994). The sketch at the top left in fig.4.h shows the concentration of CYN in seawater as a function of time and toxicity (left). Whereas the image at the bottom right of the same figure displays the amounts of CYN in fronds collected monthly at 5m depth in Cap Martin (France) in relation to the water temperature. On the other hand, the image at the bottom left reveals CYN concentrations as a function of the season and superimposed the inhibition of sea urchin egg cleavage in the methanolic extracts of C.taxifolia fronds (collected monthly at 5m depth at Cap Martin, France). This inhibitory effect is pictured in the top right part of figure 4.h. Even if CYN appears to be broken down in seawater, thus limiting its action to close-range vicinity of the algae, the degraded products are toxic and may influence the planktonic compartment as well as larval recruitments. In fact CYN is known for its repulsive and antifouling effects (4.54Meyer and Paul, 1992) confirming the presence of highest CYN at the surface of the alga with a negative gradient in the immediate seawater environment (4.51Amade & Lemée, 1998).


Fig.4.h Caulerpenyne concentrations and P.lividus mortality (75kB)

C.taxifolia protects itself by producing substances that are toxic to the Mediterranean's two main macro-herbivores, sea urchins and their eggs (besides those of hamsters and mice tested in the lab) (4.43Lemée et al., 1993), and the herbivorous fish Sarpa salpa. Data collected provide evidence that CYN:
(1) exhibits antiproliferative activity in bacterial cultures of Planococcus (4.55Giannotti et al., 1994);
(2) toxic to ciliates (4.56Dini et al., 1994);
(3) inhibits first cleavage of sea urchin eggs without affecting fertilization by reducing cytosolic ATP-dependent Ca2+ accumulation;
(4) blocks the S-phase and mitotic cycle of sea urchin embryos at metaphase by modifying microtubule networks (4.57Barbier et al., 2001) and inhibiting the stimulation of mitogen-activated protein kinase / phosphorylation (4.41Pesando et al., 1996);
(5) while Fischel et al., (4.581995) and Barbier et al., (4.572001) reported about the antiproliferative as well as growth-inhibitory effects of sesquiterpene in eight cancer cell lines of human origin (similar results have been obtained with in vitro tests of caulerpin by Ayyad & Badria (4.401994));
(6) CYN is known to induces neurological disorders (i.e. amnesia, vertigo, and hallucinations, reported by 4.59De Haro et al., 1993) on patients with food poisoning due to the ingestion of Sarpa salpa that fed on C.taxifolia);
(7) Brunelli et al., (4.602000) and Barbier et al., (4.572001) further found that CYN, besides its inhibitory effect on the Na+/K+-ATPase, also affects some other ion channels accounting for reduced after-hyperpolarization amplitudes and the decrease of cellular membrane resistance;
(8) Schröder et al., (4.611998) have shown that multixenobiotic resistance (MXR) membrane pumps - present in marine organisms - are negatively affected by CYN and caulerpin; i.e. otherwise non-lethal concentrations of environmental toxins in combination with suppressed MXR mechanism resulted in a strong apoptotic response of target cells.

As species-poor meadows of C.taxifolia cover the infralittoral zone, it replaced the rich natural algal populations along with the disappearance of many of the normally occurring species associated with it, resulting in a drastic reduction in the richness and diversity of the Mediterranean littoral ecosystem. Due to the synthesis of toxic secondary metabolites (mono- and sesqui-terpenes) C.taxifolia has another advantage over the native seaweeds and sea-grasses. Eventually, the effect of the aquarium strain C.taxifolia in the Mediterranean is a major ecological event not only by protecting itself from predation (4.14Boudouresque et al., 1995), but also by significantly decreasing species diversity and fish biomass when compared with the P.oceanica beds. All this suggests that coastal ecosystems are clearly under threat, which will result in total uniformisation of underwater landscapes and their associated populations.

Please continue with PART V - Control Measures, Strategies, Prevention, and Conclusion