Learning takes time: Biotic resistance by native herbivores increases through the invasion process.

As invasive species spread, the ability of local communities to resist invasion depends on the strength of biotic interactions. Evolutionarily unused to the invader, native predators or herbivores may be initially wary of consuming newcomers, allowing them to proliferate. However, these relationships may be highly dynamic, and novel consumer-resource interactions could form as familiarity grows. Here, we explore the development of effective biotic resistance towards a highly invasive alga using multiple space-for-time approaches. We show that the principal native Mediterranean herbivore learns to consume the invader within less than a decade. At recently invaded sites, the herbivore actively avoided the alga, shifting to distinct preference and high consumptions at older sites. This rapid strengthening of the interaction contributed to the eventual collapse of the alga after an initial dominance. Therefore, our results stress the importance of conserving key native populations to allow communities to develop effective resistance mechanisms against invaders.

Seasonal Dynamics of Epiphytic Microbial Communities on Marine Macrophyte Surfaces.

Surfaces of marine macrophytes are inhabited by diverse microbial communities. Most studies focusing on epiphytic communities of macrophytes did not take into account temporal changes or applied low sampling frequency approaches. The seasonal dynamics of epiphytic microbial communities was determined in a meadow of Cymodocea nodosa invaded by Caulerpa cylindracea and in a monospecific settlement of C. cylindracea at monthly intervals. For comparison the ambient prokaryotic picoplankton community was also characterized. At the OTU level, the microbial community composition differed between the ambient water and the epiphytic communities exhibiting host-specificity. Also, successional changes were observed connected to the macrophyte growth cycle. Taxonomic analysis, however, showed similar high rank taxa (phyla and classes) in the ambient water and the epiphytic communities, with the exception of Desulfobacterota, which were only found on C. cylindracea. Cyanobacteria showed seasonal changes while other high rank taxa were present throughout the year. In months of high Cyanobacteria presence the majority of cyanobacterial sequences were classified as Pleurocapsa. Phylogenetic groups present throughout the year (e.g., Saprospiraceae, Rhodobacteraceae, members without known relatives within Gammaproteobacteria, Desulfatitalea, and members without known relatives within Desulfocapsaceae) constituted most of the sequences, while less abundant taxa showed seasonal patterns connected to the macrophyte growth cycle. Taken together, epiphytic microbial communities of the seagrass C. nodosa and the macroalga C. cylindracea appear to be host-specific and contain taxa that undergo successional changes.

Foraging of the sea urchin Paracentrotus lividus (Lamarck, 1816) on invasive allochthonous and autochthonous algae.

Attempts to control marine invasive alien species (IAS) with native predators gained contrasting results, so far. To explore the feasibility of this approach to control the invasive marine alga Caulerpa cylindracea, we investigated the foraging behaviour of the sea urchin Paracentrotus lividus on three native macroalgae (Ulva sp., Penicillus capitatus and Cystoseira compressa) and on C. cylindracea. The consumption rate of C. cylindracea fresh biomass resulted larger than that of the other algae, when offered separately or in combination. C. cylindracea, however, was not the most attractive food item. The larger consumption rates of C. cylindracea can be explained by its specific caloric content (as assessed by its biochemical composition) that is lower than that of the other algae. Our results confirm that P. lividus can feed on C. cylindracea, but do not fully support its use to control C. cylindracea, unless in conditions where this alga is largely dominant because of other factors.

The role of competition and herbivory in biotic resistance against invaders: a synergistic effect.

Invasive species pose a major threat to global diversity, and once they are well established their eradication typically becomes unfeasible. However, certain natural mechanisms can increase the resistance of native communities to invaders and can be used to guide effective management policies. Both competition and herbivory have been identified as potential biotic resistance mechanisms that can limit plant invasiveness, but it is still under debate to what extent they might be effective against well-established invaders. Surprisingly, whereas biotic mechanisms are known to interact strongly, most studies to date have examined single biotic mechanisms separately, which likely influences our understanding of the strength and effectiveness of biotic resistance against invaders. Here we use long-term field data, benthic assemblage sampling, and exclusion experiments to assess the effect of native assemblage complexity and herbivory on the invasion dynamics of a successful invasive species, the alga Caulerpa cylindracea. A higher complexity of the native algal assemblage limited C. cylindracea invasion, probably through competition by canopy-forming and erect algae. Additionally, high herbivory pressure by the fish Sarpa salpa reduced C. cylindracea abundance by more than four times. However, long-term data of the invasion reflects that biotic resistance strength can vary across the invasion process and it is only where high assemblage complexity is concomitant with high herbivory pressure, that the most significant limitation is observed (synergistic effect). Overall, the findings reported in this study highlight that neglecting the interactions between biotic mechanisms during invasive processes and restricting the studied time scales may lead to underestimations of the true capacity of native assemblages to develop resistance to invaders.

Selective DNA and Protein Isolation From Marine Macrophyte Surfaces.

Studies of unculturable microbes often combine methods, such as 16S rRNA sequencing, metagenomics, and metaproteomics. To apply these techniques to the microbial community inhabiting the surfaces of marine macrophytes, it is advisable to perform a selective DNA and protein isolation prior to the analysis to avoid biases due to the host material being present in high quantities. Two protocols for DNA and protein isolation were adapted for selective extractions of DNA and proteins from epiphytic communities inhabiting the surfaces of two marine macrophytes, the seagrass Cymodocea nodosa and the macroalga Caulerpa cylindracea. Protocols showed an almost complete removal of the epiphytic community regardless of the sampling season, station, settlement, or host species. The obtained DNA was suitable for metagenomic and 16S rRNA sequencing, while isolated proteins could be identified by mass spectrometry. Low presence of host DNA and proteins in the samples indicated a high specificity of the protocols. The procedures are based on universally available laboratory chemicals making the protocols widely applicable. Taken together, the adapted protocols ensure an almost complete removal of the macrophyte epiphytic community. The procedures are selective for microbes inhabiting macrophyte surfaces and provide DNA and proteins applicable in 16S rRNA sequencing, metagenomics, and metaproteomics.

A new record of the invasive seaweed Caulerpa cylindracea Sonder in the South Adriatic Sea.

The green alga Caulerpa cylindracea Sonder is one of the most infamous and threatening invasive species in the Mediterranean Sea. Since 1985, it started rapidly spreading to all Mediterranean regions causing many ecological changes on natural communities. In the present study, we present an example of this proliferation with the first record in the Marine Protected Area of Tremiti Island (MPATI) in the South Adriatic Sea. Fifteen sites along the coast and 5 different depths have been investigated. Our results provide eveidence of a wide invasion of this pest in three islands, San Domino, San Nicola and Capraia. This study fills a particular data gap in the ongoing biomonitoring of invasive seaweeds in the Mediterranean Sea representing a base line of this invasive species for the MPATI.

Negative effects of warming on seagrass seedlings are not exacerbated by invasive algae.

The observed and projected rise in sea surface temperature challenges marine biodiversity worldwide, and particularly in temperate ecosystems dealing with the arrival of novel species of tropical provenance. When the impacted biota are early life stages of ecosystem engineers, the effects of those impacts are of major concern for ecologists and coastal managers. We experimentally examined the individual and potential additive effects of seawater warming and the presence of the invasive algae on the development of seedlings of the seagrass Posidonia oceanica in a three-month mesocosm experiment. Whereas the presence of the invasive algae (Caulerpa cylindracea and Lophocladia lallemandii) did not result in detrimental effects on seedlings, warming negatively affected seedling development. Interestingly, the presence of both invasive algae may ameliorate the negative effects of warming.

Effect of the algal alkaloid caulerpin on neuropeptide Y (NPY) expression in the central nervous system (CNS) of Diplodus sargus.

Recent studies have suggested that Mediterranean indigenous fish species are affected by bioactive metabolites coming from marine invasive species via food web interactions. In particular, both physiological and behavioural changes in the white sea bream Diplodus sargus were related to caulerpin (CAU), a bisindolic alkaloid particularly abundant in the invasive alga Caulerpa cylindracea, on which the fish actively feed. Dietary administration of CAU decreased aggressiveness in D. sargus, suggesting an anxiolytic-like effect of CAU possibly mediated by endogenous anxiolytic agents. This hypothesis is supported here by the finding of a significant increase of NPY transcriptional expression in the brain of fish fed with CAU enriched food, shedding more light on the neural mechanisms behind the altered behaviour of D. sargus.

Fishing for Targets of Alien Metabolites: A Novel Peroxisome Proliferator-Activated Receptor (PPAR) Agonist from a Marine Pest.

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)-a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin-is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

¹H NMR Spectroscopy and MVA to Evaluate the Effects of Caulerpin-Based Diet on Diplodus sargus Lipid Profiles.

The biological invasion of the green algae Caulerpa cylindracea represents a serious scientific and public issue in the Mediterranean Sea, essentially due to strong modifications both to habitat structure and native benthic communities. Although alterations in health status and changes in flesh quality of some marine species (dietary exposed to C. cylindracea) have been observed, no studies on cause-effect relationships have been carried out. Here, for the first time, through a controlled feeding experiment followed by ¹H NMR Spectroscopy and multivariate analysis (PCA, OPLS-DA), we showed that caulerpin taken with diet is directly responsible of changes observed in metabolic profile of fish flesh, including alteration of lipid metabolism, in particular with a reduction of ω3 PUFA content. The potential of caulerpin to directly modulate lipid metabolism opens up new questions about causal mechanism triggered by algal metabolite also in view of a possible exploitation in the nutraceutical/medical field.

Propagules are not all equal: traits of vegetative fragments and disturbance regulate invasion success.

Invasion success is regulated by multiple factors. While the roles of disturbance and propagule pressure in regulating the establishment of non-native species are widely acknowledged, that of propagule morphology (a proxy for quality) is poorly known. By means of a multi-factorial field experiment, we tested how the number (5 vs. 10) and quality (intact, without fronds or without rhizoids) of fragments of the clonal invasive seaweed, Caulerpa cylindracea, influenced its ability to establish in patches of the native seagrass, Posidonia oceanica, exposed to different intensities of disturbance (0, 50, or 100% reduction in canopy cover). We hypothesized that the ability of fragments to establish would be greater for intact fragments (high quality) and reduced more by frond removal (low quality) than rhizoid removal (intermediate quality). At low propagule pressure or quality, fragment establishment was predicted to increase with increasing disturbance, whereas, at high propagule pressure or quality, it was predicted to be high regardless of disturbance intensity. Disturbance intensity, fragment number and quality had independent effects on C. cylindracea establishment success. Disturbance always facilitated fragment establishment. However, fragments retaining fronds, either intact or deprived of rhizoids, had higher establishment success than fragments deprived of fronds. Increasing propagule number had weak effects on the cover of C. cylindracea. Our results demonstrate that propagule traits enabling the acquisition of resources made available by disturbance can be more important than propagule number in determining the establishment and spread of clonal non-native plants. More generally, our study suggests that propagule quality is a key, yet underexplored, determinant of invasion success.

Eutrophication affects the resistance of fucoids to an introduced alga spread.

This study investigates whether eutrophication can affect the capacity of the canopy alga Cystoseira brachycarpa to impede or limit the spread of the introduced species Caulerpa cylindracea. By means of a manipulative field study (16 months long), the effects of nutrient enrichment and C. cylindracea removal were tested on the canopy-alga and the associated macroalgal community. Results highlighted deep changes through time due to nutrient enrichment, as C. brachycarpa decreased and Halopteris scoparia increased in cover. Furthermore, C. brachycarpa was also affected by the presence of the introduced species Caulerpa cylindracea which, in turn, was found significantly advantaged by nutrient enrichment. Overall, our findings suggest that eutrophication can drive the substitution of Cystoseira with H. scoparia, leading to the shift from canopy to opportunistic species, which are unable to avoid the spread of C. cylindracea.

Carry over effects of nutrient addition on the recovery of an invasive seaweed from the winter die-back.

Nutrient enrichment of coastal waters can enhance the invasibility and regrowth of non-native species. The invasive alga Caulerpa cylindracea has two distinct phases: a well-studied fast-growing summer phase, and a winter latent phase. To investigate the effects of nutrient enrichment on the regrowth of the seaweed after the winter resting-phase, a manipulative experiment was carried out in intertidal rockpools in the North-western Mediterranean. Nutrients were supplied under different temporal regimes: press (constant release from January to May), winter pulse (January to March) and spring pulse (March to May). Independently from the temporal characteristics of their addition, nutrients accelerated the re-growth of C. cylindracea after the winter die-back, resulting in increased percentage covers at the peak of the growing season. Nutrient addition did not influence the number and length of fronds and the biomass. Native components of the algal community did not respond to nutrient additions. Our results show that nutrient supply can favour the spread of C. cylindracea even when occurring at a time of the year at which the seaweed is not actively growing.

Trophic interactions between indigenous and non-indigenous species in Lampedusa Island, Mediterranean Sea.

Using stable isotope analysis, we investigated trophic interactions between indigenous benthic taxa and the non-indigenous species (NIS): the green alga Caulerpa cylindracea, the red alga Asparagopsis taxiformis, the crab Percnon gibbesi and the sea hare Aplysia dactylomela. The study was conducted on Lampedusa Island, Mediterranean Sea. We evaluated the trophic positions and isotopic niches of consumers. Using Bayesian mixing models, we quantified the food source contribution to diets of indigenous and non-indigenous herbivores. Isotopic niche of NIS showed no overlap with the ones of indigenous macroinvertebrates and fish. Caulerpa cylindracea provided the largest contribution to the diet of P. gibbesi (0.431-1), while the dietary contribution estimates overlapped considerably for all sources of A. dactylomela and indigenous herbivores. From these results, we conclude that the invasion of C. cylindracea is increasing the diversity of available prey and might facilitate the expansion of other NIS.

Meiofauna communities, nematode diversity and C degradation rates in seagrass (Posidonia oceanica L.) and unvegetated sediments invaded by the algae Caulerpa cylindracea (Sonder).

We investigated meiofauna and sedimentary C cycling in seagrass (Posidonia oceanica) and unvegetated sediments invaded and not invaded by the non-indigenous tropical algae Caulerpa cylindracea. In both habitats, invaded sediments were characterized by higher organic matter contents. No effect was observed for prokaryotes and C degradation rates. In seagrass sediments, C turnover in invaded beds was about half that in not invaded ones. Meiofaunal communities varied significantly among invaded and not invaded grounds only in bare sediments. In both habitats, nematode species richness and assemblage composition were not affected by the algae. The effect of C. cylindracea on the turnover and nestedness components of the Jaccard dissimilarity varied between the two habitats. We show that the presence of C. cylindracea gives rise to variable consequences on meiofauna biodiversity and C cycling in different habitats. We conclude that further studies across different habitats and ecological components are needed to ultimately understand and predict the consequences of C. cylindracea invasion in shallow Mediterranean ecosystems.

Linking disturbance and resistance to invasion via changes in biodiversity: a conceptual model and an experimental test on rocky reefs.

Biological invasions threaten biodiversity worldwide. Nonetheless, a unified theory linking disturbance and resistance to invasion through a mechanistic understanding of the changes caused to biodiversity is elusive. Building on different forms of the disturbance-biodiversity relationship and on the Biotic Resistance Hypothesis (BRH), we constructed conceptual models showing that, according to the main biodiversity mechanism generating invasion resistance (complementary vs. identity effects), disturbance can either promote or hinder invasion. Following the Intermediate Disturbance Hypothesis (IDH), moderate levels of disturbance (either frequency or intensity) are expected to enhance species richness. This will promote invasion resistance when complementarity is more important than species identity. Negative effects of severe disturbance on invasion resistance, due to reductions in species richness, can be either overcompensated or exacerbated by species identity effects, depending on the life-traits becoming dominant within the native species pool. Different invasion resistance scenarios are generated when the diversity-disturbance relationship is negative or positive monotonic. Predictions from these models were experimentally tested on rocky reefs. Macroalgal canopies differing in species richness (1 vs. 2 vs. 3) and identity, were exposed to either a moderate or a severe pulse disturbance. The effects of different canopy-forming species on the seaweed, Caulerpa cylindracea, varied from positive (Cystoseira crinita) to neutral (Cystoseira barbata) to negative (Cystoseira compressa). After 2 years, severely disturbed plots were monopolized by C. compressa and supported less C. cylindracea. Our study shows that the effects of disturbance on invasion depend upon its intensity, the main mechanism through which biodiversity generates invasion resistance and the life-traits selected within the native species pool. Disturbance can sustain invasion resistance when promoting the dominance of competitively subordinate species possessing traits that allow outperforming invaders.

The effects of an invasive seaweed on native communities vary along a gradient of land-based human impacts.

The difficulty in teasing apart the effects of biological invasions from those of other anthropogenic perturbations has hampered our understanding of the mechanisms underpinning the global biodiversity crisis. The recent elaboration of global-scale maps of cumulative human impacts provides a unique opportunity to assess how the impact of invaders varies among areas exposed to different anthropogenic activities. A recent meta-analysis has shown that the effects of invasive seaweeds on native biota tend to be more negative in relatively pristine than in human-impacted environments. Here, we tested this hypothesis through the experimental removal of the invasive green seaweed, Caulerpa cylindracea, from rocky reefs across the Mediterranean Sea. More specifically, we assessed which out of land-based and sea-based cumulative impact scores was a better predictor of the direction and magnitude of the effects of this seaweed on extant and recovering native assemblages. Approximately 15 months after the start of the experiment, the removal of C. cylindracea from extant assemblages enhanced the cover of canopy-forming macroalgae at relatively pristine sites. This did not, however, result in major changes in total cover or species richness of native assemblages. Preventing C. cylindracea re-invasion of cleared plots at pristine sites promoted the recovery of canopy-forming and encrusting macroalgae and hampered that of algal turfs, ultimately resulting in increased species richness. These effects weakened progressively with increasing levels of land-based human impacts and, indeed, shifted in sign at the upper end of the gradient investigated. Thus, at sites exposed to intense disturbance from land-based human activities, the removal of C. cylindracea fostered the cover of algal turfs and decreased that of encrusting algae, with no net effect on species richness. Our results suggests that competition from C. cylindracea is an important determinant of benthic assemblage diversity in pristine environments, but less so in species-poor assemblages found at sites exposed to intense disturbance from land-based human activities, where either adverse physical factors or lack of propagules may constrain the number of potential native colonizers. Implementing measures to reduce the establishment and spread of C. cylindracea in areas little impacted by land-based human activities should be considered a priority for preserving the biodiversity of Mediterranean shallow rocky reefs.

1H NMR Spectroscopy and MVA Analysis of Diplodus sargus Eating the Exotic Pest Caulerpa cylindracea.

The green alga Caulerpa cylindracea is a non-autochthonous and invasive species that is severely affecting the native communities in the Mediterranean Sea. Recent researches show that the native edible fish Diplodus sargus actively feeds on this alga and cellular and physiological alterations have been related to the novel alimentary habits. The complex effects of such a trophic exposure to the invasive pest are still poorly understood. Here we report on the metabolic profiles of plasma from D. sargus individuals exposed to C. cylindracea along the southern Italian coast, using 1H NMR spectroscopy and multivariate analysis (Principal Component Analysis, PCA, Orthogonal Partial Least Square, PLS, and Orthogonal Partial Least Square Discriminant Analysis, OPLS-DA). Fish were sampled in two seasonal periods from three different locations, each characterized by a different degree of algal abundance. The levels of the algal bisindole alkaloid caulerpin, which is accumulated in the fish tissues, was used as an indicator of the trophic exposure to the seaweed and related to the plasma metabolic profiles. The profiles appeared clearly influenced by the sampling period beside the content of caulerpin, while the analyses also supported a moderate alteration of lipid and choline metabolism related to the Caulerpa-based diet.

High tolerance to simulated herbivory in the clonal seaweed, Caulerpa cylindracea.

Tolerance can enable introduced plants to establish in the face of intense consumption by native herbivores. The siphonacous seaweed, Caulerpa cylindracea, despite being heavily grazed by native herbivores, is one of the most successful invaders in the Mediterranean. By means of a field experiment simulating herbivore grazing, we tested whether regeneration from damaged creeping stolons may allow C. cylindracea to compensate or overcompensate for biomass loss. In order to simulate different grazing intensities, the cover of C. cylindracea was either left untouched or clipped to 25%, 50% or 75% of the original value. After 2 months, C. cylindracea cover increased by ∼ 450% in 75% removal plots, ∼ 200% in 50% removals and ∼ 70% in 25% removals, whilst the increment in controls was just ∼ 6%. Such differential growth rates resulted in no difference in the cover of C. cylindracea between clipped (irrespective of clipping intensity) and control plots. Thus, regeneration from remnant clipped stolons could compensate for biomass loss, suggesting that non-native siphonaceous seaweeds can withstand intense mechanical damage and, possibly, grazing by herbivores. This compensatory mechanism may underpin the success of some of the most invasive clonal plants.

Spreading patterns of the invasive Caulerpa cylindracea Sonder along the west Istrian Coast (northern Adriatic Sea, Croatia).

The northern Adriatic Sea represents the northernmost and thus the coldest biogeographic sector of the Mediterranean Sea. In 2004, the invasive green alga Caulerpa cylindracea was recorded for the first time in the northern Adriatic at a site of the west Istrian Coast. Until 2010, additional C. cylindracea mats have only formed up to 7 km northward from the first colonisation site. Subsequently, the alga was also recorded at sites widespread along the entire coast. Both the first 2004 colonisation event and the 2011-2014 colonisation of distant sites occurred during periods of winter seawater temperatures higher than 9 °C. In general, algal spreading was markedly slow. Approximately 10 years after the first record, C. cylindracea has affected less than 1% of the entire west Istrian coastline. The colonisation predominantly occurred in ports and urbanised bays (seaside resorts) suggesting that anthropogenic activities might enhance algal diffusion.