Antibacterial scalarane from Doriprismatica stellata nudibranchs (Gastropoda, Nudibranchia), egg ribbons, and their dietary sponge Spongia cf. agaricina (Demospongiae, Dictyoceratida)

Investigations on the biochemical relationship between Doriprismatica stellata (Chromodorididae, Doridoidea) nudibranchs, their egg ribbons, and the associated dietary sponge Spongia cf. agaricina (Demospongiae, Porifera) led to the isolation of the structurally new scalarane-type sesterterpene 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin, with an unprecedented position of the cyclopropane ring annelated to the ring A. Unlike other scalaranes, which are most often functionalized at C-12 of ring C, it bears two acetoxy groups at C-11 and C-24 instead. The compound was present in all three samples, supporting the dietary relationship between chromodorid nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the Spongiidae family. The results also indicate that D. stellata passes the scalarane metabolite on to its egg ribbons, most likely for protective purposes. The scalarane showed antibacterial activity against the Gram-positive bacteria Arthrobacter crystallopoietes (DSM 20117) and Bacillus megaterium (DSM 32).


Results
Chemical investigation on Doriprismatica stellata nudibranchs, egg ribbons and Spongia cf. agaricina The new molecule was isolated as a white amorphous solid from D. stellata nudibranchs (11 mg, 0.3% wet weight). Specific optical rotation was measured in chloroform (c = 0.6), giving [α] D +40.5. The molecular formula C 29 H 42 O 6 was established based on 13 C NMR data and HRAPCIMS measurements, yielding m/z 487.3054 [M + H] + (Supporting Information File 1). The double bond equivalent (DBE) was calculated to be nine and together with the 13 C NMR data, giving evidence for one C-C and two C-O double bonds, thus suggested a structure Table 1: NMR spectroscopic data of 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin (CDCl 3 ).   Figure S8, Supporting Informa-  The analysis of the 2D NMR data and comparison to literature values [53] suggested that the compound belongs to the family of scalarane sesterterpenoids, with similarities to the deoxoscalarin-like molecule 12,24-diacetoxydeoxoscalarin, previously isolated from a Korean sponge of the genus Spongia [53]. The two acetoxy groups were located at the C-11 (δ 68. The entire assignment of all NMR data is given in Table 1. The relative configuration was determined from proton coupling constants and NOE data (Table 1, Figure 4). the structure and relative configuration of 12-deacetoxy-4demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin was determined. It needs to be noted that the molecule was unstable over time, especially in ring E, and a variety of degradation products formed by, inter alia, hydrolysis of the hemiacetal and loss of the acetoxy groups.
The new scalarane was also detected in Doriprismatica stellata egg ribbons and Spongia cf. agaricina (Figure 3). It was isolated from both samples (egg ribbons: 1 mg, 0.1% wet weight; sponge: 0.7 mg, 0.02% wet weight) and the identity was validated by comparison of the MS and NMR spectra.
Primordially, chromodorid nudibranchs feed upon a broad range of sponges, however, more derived genera like Glossodoris and Doriprismatica have taken to feeding upon a narrow range of sponges [23,27,47]. As the first chemical investigation of D. stellata nudibranchs, our results indicate that these sea slugs live and feed upon the dictyoceratid sponge Spongia cf.
agaricina. This, among other investigations on Doriprismatica atromarginata [38,41,44,45,50] and D. sedna [39], supports the idea of a stenophagous dietary relationship between nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the families Thorectidae and Spongiidae. This relationship is further reflected by their shared specialized metabolite 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin, as proven in this study. Sesterterpenes are a rare terpene class, accounting for less than 2% of all known terpenoids, with only a few reports on their biosynthesis [72][73][74][75][76]. However, their frequent occurrence in marine organisms is striking and sponges are considered as the prime source of these terpenoids [25]. Yet determining the origin and in vitro production of these metabolites is anything but trivial. Sponges are known to host complex symbiont communities, with up to 30-60% as microbial biomass [13,77]. These highly species-specific communities are most probably vertically transmitted [78] and were shown to share and cover various core functions of sponge metabolism by functionally equivalent symbionts, analogous enzymes, or biosynthetic pathways [16,79,80]. Another Spongia species, S. officinalis, was shown to harbour bacteria with terpenoid cyclases/protein prenyltransferases responsible for a wide chemodiversity of terpenoid natural products [14,81]. Besides, the marine fungi Penicillium spp. and Aspergillus spp. are often associated with sponge hosts and were found to produce various terpenoids as well [15,82,83]. Hence, if sponges are not the origin of these metabolites, it is tempting to argue that the sesterterpene biosynthesis could be performed or mediated by their microbial symbionts. This further indicates a close association, interconnectedness, and probable co-evolution between microorganisms, sponges and nudibranchs [9]. D. stellata was not only found to sequester and accumulate 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin from Spongia cf.
agaricina, but to pass it on to the egg ribbons as well. This, in addition to its bioactivity, might suggest a biological role, either as protection against predation, fouling, or in the reproductive cycle, as mentioned in previous studies on nudibranch egg ribbons [17,[34][35][36]. The antibacterial activity of 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin could point towards a potential protective role against bacterial biofilm formation. Unfortunately, the metabolite was unstable over time and it was not possible to conduct further assays. Future studies on scalarane sesterterpenes could reveal their full potential and true biological and ecological functions in complex, co-evolved communities.

Experimental
General experimental procedures

Extraction and isolation
Six Doriprismatica stellata nudibranchs (3.7 g wet weight), their egg ribbons (0.7 g wet weight) and pieces of the associated sponge (3.5 g wet weight) were separately frozen, crushed and ultrasonicated for a total of 3 minutes (30 s intervals) on ice, while submerged in a minimum of first acetone (Ac) and consecutively methanol (MeOH). The ethanolic storage solutions of D. stellata nudibranch, egg ribbon, and Spongia cf. agaricina samples were each combined with the respective Ac/MeOH extracts of the samples and dried under vacuum to give the crude extracts. After liquid-liquid separation of the three crude extracts (0.9 g, 0.3 g, and 0.2 g, respectively) between 50