Metabolomic profiling reveals deep chemical divergence between two morphotypes of the zoanthid Parazoanthus axinellae

Abstract

Metabolomics has recently proven its usefulness as complementary tool to traditional morphological and genetic analyses for the classification of marine invertebrates. Among the metabolite-rich cnidarian order Zoantharia, Parazoanthus is a polyphyletic genus whose systematics and phylogeny remain controversial. Within this genus, one of the most studied species, Parazoanthus axinellae is prominent in rocky shallow waters of the Mediterranean Sea and the NE Atlantic Ocean. Although different morphotypes can easily be distinguished, only one species is recognized to date. Here, a metabolomic profiling approach has been used to assess the chemical diversity of two main Mediterranean morphotypes, the "slender" and "stocky" forms of P. axinellae. Targeted profiling of their major secondary metabolites revealed a significant chemical divergence between the morphotypes. While zoanthoxanthin alkaloids and ecdysteroids are abundant in both morphs, the "slender" morphotype is characterized by the presence of additional and bioactive 3,5-disubstituted hydantoin derivatives named parazoanthines. The absence of these specific compounds in the "stocky" morphotype was confirmed by spatial and temporal monitoring over an annual cycle. Moreover, specimens of the "slender" morphotype are also the only ones found as epibionts of several sponge species, particularly Cymbaxinella damicornis thus suggesting a putative ecological link.

Figure 1. The two morphotypes of the Mediterranean zoanthid Parazoanthus axinellae studied here.

A) “Stocky” morphotype growing in dense population, directly on the rocky substratum; B) “Slender” morphotype growing on the sponge Cymbaxinella damicornis; and C) Savalia savaglia growing on the gorgonian Paramuricea clavata (Pictures from T. Pérez).

Figure 2. The three families of compounds isolated from Parazoanthus axinellae.

Figure 3. Map of the Western Mediterranean with Parazoanthus axinellae sampling locations indicated as black circles (see details in Table 1).

Black square represents the site (Mo1) where a 1-yr time series was conducted. Black star represents the sampling location for the outgroup Savalia savaglia (Marseille). This map was obtained from http://d-maps.com/carte.php?num_car=3122&lang=fr (Date of access: 16 Apr. 2013) and modified under Adobe Illustrator.

Figure 4. Principal Component Analysis (PCA) of metabolomic profiles of both P. axinellae morphotypes (17 “slender” and 9 “stocky” morphotypes) according to the location (score plots).

Figure 5. Principal Component Analysis (PCA) of metabolomic profiles of both P. axinellae morphotypes (“slender” and “stocky”) according to the location (loading plots).

Figure 6. Temporal variation (one annual cycle) of three target metabolite families for the “stocky” and “slender” morphotypes.

Metabolite expression level is displayed as peak area in the Base Peak Chromatogram (no data are available for the stocky morphotype on the 13^th January 2009).

Figure 7. Bioactivity assay (Microtox®) performed on extracts and pure compounds from P. axinellae.