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Ayuningrum D, Kristiana R, Nisa AA, Radjasa SK, Muchlissin SI, Radjasa OK, Sabdono A, Trianto A. 2019. Bacteria associated with tunicate, Polycarpa aurata, from Lease Sea, Maluku, Indonesia exhibiting anti-multidrug resistant bacteria. Biodiversitas 20: 956-964. Tunicate is a rich secondary metabolites producer with various biological activities whether as an original producer or produced by the associated microorganisms. In this study, a total of 11 tunicate specimens were identified as Polycarpa aurata with four color variations based on morphological characteristic and COI gene identification and BLAST analysis. The P. aurata associated-bacteria were isolated and tested for antimicrobial activity against multi-drug resistant (MDR) bacteria. A total of 86 axenic isolates were successfully purified. Furthermore, nine isolates (10.5%) exhibited antibacterial activity on preliminary screening. Nine prospective isolates were fermented in respective medium (Zobell 2216, modified M1 or modified ISP2 media) then extracted using ethyl acetate. The ethyl acetate extracts from liquid fermentation were tested against MDR Escherichia coli, MDR Bacillus cereus, Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Sensitive and Staphylococcus aureus (MSSA). As a result, seven isolates (8.1%) still retained the activity at the extract concentration 150 µg/disk. Molecular analysis based on 16S rDNA sequencing revealed the most active isolates, TSB 47, TSC 10 and TSB 34 identified as Bacillus tropicus, Vibrio alginolyticus and Virgibacillus massiliensis, with BLAST homology 99%.

Small island archipelagos with fringing and dispersed reef systems represent special marine ecosystems, providing a patchy habitat for many coral reef organisms. Although geographic distances may be short, it is still unclear if such environments are inhabited evenly with panmictic conditions or if limited connectivity between marine populations, even on small geographic scales, leads to genetic differentiation between areas within the archipelago or even single reef structures. To study diversity patterns and connectivity between reefs of the Spermonde Archipelago, Indonesia, population genetic analyses of two reef organisms were performed by using the mitochondrial control region and microsatellite markers. A vertebrate (clown anemonefish) and an invertebrate species (sea squirt) were studied in parallel to investigate if there are general patterns of connectivity in Spermonde for sessile or site attached marine species, which can be extrapolated to a larger group. The genetic population structures revealed restrictions in gene flow in the clown anemone fish (Amphiprion ocellaris), especially between near-shore reefs in the South of the archipelago. This indicates very localized genetic exchange and may also reflect the high self-recruitment typical for these fish. The northern reefs show higher connectivity despite geographic distances being larger. The filter-feeding sessile sea squirt, Polycarpa aurata, features similar population patterns, especially in the southern area. However, connectivity is generally higher in the middle and shelf edge areas of Spermonde for this species. The results underline that there are restrictions to gene flow even on very small geographic scales in the studied organisms, with many barriers to gene flow in the southern shallower shelf area. Weaker currents in this area may lead to more influence of biological factors for dispersal, such as larval behavior, motility and competition for suitable habitat. The results are discussed in the context of conservation and MPA planning in the Spermonde Archipelago.

N,N-Didesmethylgrossularine-1 (DDMG-1) has a rare β-carboline structure and was isolated from an Indonesian ascidian Polycarpa aurata as an active component against tumor necrosis factor (TNF)α production in lipopolysaccharine (LPS)-stimulated murine macrophase-like RAW 264.7 cells as reported in our previous paper. Further investigation on the inhibitory activity of DDMG-1 against the production of inflammatory cytokines in human monocytic THP-1 cells, we found that DDMG-1 reduced the excess production of IL-8 in LPS- stimulated THP-1 cells through inhibition of the mRNA level of IL-8 1κB-α degradation, and binding of NF-xfi to the target DNA site. The THP-1 cells used in this study showed the high production of IL-8 by the stimulation with TNF-α, IL-1β, and 12-O-tetradecanoylphorbol-13-acetate (PMA) as similar to LPS. DDMG-1 inhibited the IL-8 production in IL-1β-stimulated THP-1 cells but did not show an inhibitory activity in the cells stimulated by TNF-α and PMA. Therefore, DDMG-1 was specific to the IL-1β signalling pathway. These results suggested that DDMG-1 could be a useful drug candidate lead compound to control the excess production of IL-8.

A deep study of the metabolic content of the tunicate Polycarpa aurata, collected from Indonesian coast, afforded the isolation of two novel alkaloids, polyaurines A (1) and B (2), along with two new p-substituted benzoyl derivatives (3 and 4) and four known compounds (5&ndash, 8). The structural elucidation of the new secondary metabolites was assigned by 1D, 2D NMR, and HRESIMS techniques. Computational studies resulted a useful tool to unambiguously determine in polyaurine B the presence of rarely found 1,2,4-thiadiazole ring. The effects of polyaurines A and B on mammalian cells growth and on the viability of different blood-dwelling Schistosoma mansoni (phylum: Platyhelminthes) stages, as well as egg production, were evaluated. Both compounds resulted not cytotoxic, interestingly some of the eggs produced by polyaurine A-treated adult pairs in vitro are smaller, deformed, and/or fragmented, therefore, polyaurine A could represent an interesting bioactive natural molecule to be further investigated.

Two new alkaloids, polycarpathiamines A and B (1 and 2), were isolated from the ascidian Polycarpa aurata. Their structures were unambiguously determined by 1D, 2D NMR, and HRESIMS measurements and further confirmed by comparison with a closely related analogue, 3-dimethylamino-5-benzoyl-1,2,4-thiadiazole (4), that was prepared by chemical synthesis. Compounds 1 and 2 both feature an uncommon 1,2,4-thiadiazole ring whose biosynthetic origin is proposed. Compound 1 showed significant cytotoxic activity against L5178Y murine lymphoma cells (IC50 0.41 μM).

The Banda Sea region is situated in the Wallacea biogeographical area, known as one of the biodiversity hotspots of the world. The region harbours various types of ecosystems, ranging from mangroves and coral reefs to seaweeds, indicating its potential for harbouring organisms that produce bioactive compounds, such as sea cucumbers, soft corals, and tunicates. While many studies have been carried out on the first two organisms, much lesser is known about the latter one, notably in this region. The assessment is our first attempt to explore the tunicates found in northern coastal waters of Ambon Island, an area that is a part of the Banda Sea. Two types of tunicates were obtained during the survey, namely the violet-green and white-yellow variants of solitary tunicates Polycarpa aurata (15 specimens) and social ones of Didemnum molle (3 specimens). The Polycarpa specimens' heights ranged from 2.5 to 4.8 cm with wet weights from 9.4 to 28.03 g, and the Didemnum ones were of 1.2 - 2 cm and wet weights from 1.2 to 5 g. Feeding experiment indicated that low volume (5-10 ml) of pure cultures such as Dunaliella sp., Nannochloropsis sp. and Tetraselmis sp. in small aquarium (40 x 60 cm) resulted in good survival rate for Polycarpa aurata collected from northern Ambon Island waters. Rearing experiments in a discontinuous aquarium setting using solitary tunicates indicated the following requirements: thorough cleaning of the tanks, regular feeding intervals, and regular cleaning of each tunicate. The regular cleaning of mucous at the external part of tunicates is indispensable to ensure the wellbeing of the organisms.

N,N-Didesmethylgrossularine-1 (DDMG-1), a compound with a rare alpha-carboline structure, was isolated from an Indonesian ascidian Polycarpa aurata as responsible for the observed inhibitory activity against TNF-alpha production in lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells. DDMG-1 inhibited the mRNA level of mTNF-alpha, IkappaB-alpha degradation, and binding of NF-kappaB to the target DNA site in LPS-stimulated RAW 264.7 cells. Moreover, DDMG-1 had an inhibitory effect on the production of IL-8, which is produced in CD14(+)-THP-1 cells stimulated by LPS. DDMG-1 is thus a promising drug candidate lead compound for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis.

Twenty-one microsatellite loci were developed for the tropical ascidian, Polycarpa aurata. Microsatellite enriched genomic libraries were constructed and sequenced using Pacific Biosciences single molecule real time sequencing technology. The 21 loci were characterized in 20 individuals from Alor, Indonesia. Observed levels of heterozygosity ranged from 0.30 to 1.00 with a mean of 8.2 alleles per locus. Loci PA21 and PA45 showed evidence of significant linkage disequilibrium and three loci significantly deviated from Hardy–Weinberg expectations.

Polycarpine 1 , a highly cytotoxic marine natural product, has been synthesized in three steps from p-methoxyphenacyl bromide 4 in 57% overall yield. The key reaction for construction of the symmetrically substituted disulfide linkage of polycarpine is the treatment of 2-amino-4-(4-methoxyphenyl)-1-methylimidazole 17 with S2Cl2 in acetic acid. In a similar way ten related compounds, including three thiazole analogues, have been prepared. Most of them exhibit high cytotoxic activities against an array of human cancer cell lines.

Dengue Hemorrhagic Fever (DHF) is a disease caused by virus through Aedesaegypti mosquitoes as the vector. The disease is spreading across the world andendanger and threaten human life. Measures in controlling the vector using the commonlarvaside “abate” are in adequate and less affective. The objectives of the research is tofind out larvaside extracted from three kinds of ascidian. The each ascidians (Polycarpaaurata, Didemnum molle and Rophalaea crassa) were extracted with ethanol, and thecrude extracts were subjected to larvasidal test by dissolving in water containing thelarvae. The remarkable extract activity was partition in ethyl acetate, hexane andbuthanol. The results show that extract of Polycarpa aurata has the highest activity. Theactivity of fractions show that ethyl acetate at 100 ppm reveals the highest mortality oflarvae 100% in 8 hours, followed by hexane fraction (12 hours) then buthanol fraction (18hours). All the fractions (Ethyl acetate, Hexane and Buthanol) could totality kill the larvaewithin 24 hours which is comparable to the abate.

Two new alkaloids, polycarpine (1) and N,N-didesmethylgrossularine-1 (4), have been isolated from extracts of the ascidian Polycarpa aurata collected in Chuuk, Federated States of Micronesia. Three degradation products of 1 were also isolated. The structures of 1, 2, and 4 were determined by X-ray crystallography. The dimeric disulfide 1 inhibited the enzyme inosine monophosphate dehydrogenase, but the inhibition could be reversed by addition of excess dithiothreitol suggesting that 1 reacts with sulfhydryl groups on the enzyme.

Polycarpine 1 , a highly cytotoxic marine natural product, has been synthesized in three steps from p-methoxyphenacyl bromide 4 in 57% overall yield. The key reaction for construction of the symmetrically substituted disulfide linkage of polycarpine is the treatment of 2-amino-4-(4-methoxyphenyl)-1-methylimidazole 17 with S2Cl2 in acetic acid. In a similar way ten related compounds, including three thiazole analogues, have been prepared. Most of them exhibit high cytotoxic activities against an array of human cancer cell lines.

Three new sulfur-containing alkaloids, polycarpaurines A ( 1 ), B ( 2 ), and C ( 3 ) were isolated from the tropical ascidian Polycarpa aurata collected in Indonesia, together with six known compounds ( 4 – 9 ). The structures of new compounds were assigned on the basis of their spectral data. Compounds 1 , 3 , 4 , and 8 inhibited colony formation of Chinese hamster V79 cells with EC 50 values of 6.8, 8.6, 3.8, and 10 μM, respectively. Compounds 2 and 7 showed modest activity against V79 cells (EC 50 >10 μM).

Polycarpine from ascidian Polycarpa aurata was previously found to be active against different human tumor cells. In this study, we investigated the antitumor mechanisms of polycarpine and its synthetic derivative, desmethoxyethoxy-polycarpine (dimethylpolycarpine), through the induction of apoptosis. This new knowledge regarding the proapoptotic action of polycarpine and dimethylpolycarpine should lead to a better understanding of their effects and development of a new class of anticancer drugs.Apoptosis was clearly observed by flow cytometry and Western blotting using an antibody against cleaved caspase-3 as an apoptotic marker.Polycarpines differentially activated p38 kinase, JNKs, and ERKs in JB6 Cl 41 cells. The polycarpines-induced apoptosis was decreased in cells expressing a dominant-negative mutant of JNK. Both compounds stimulated p53-dependent transcriptional activity and phosphorylation. Induction of p53-phosphorylation at serine 15 was suppressed in JNKI and JNK2 knockout cells. Furthermore, polycarpines were unable to induce apoptosis in p53-deficient MEFs in contrast to a strong induction of apoptosis in wild type MEFs, suggesting that p53 is involved in apoptosis induced by polycarpines. The p53 phosphorylation in turn was mediated by activated JNKs.These results indicate that all three MAPK signaling pathways are involved in the response of JB6 cells to treatment with polycarpines. Evidence also supports a proapoptotic role of the JNKs signaling pathway in vivo and clearly indicates that JNKs are required for phosphorylation of c-Jun, activation of p53, and subsequent apoptosis induced by polycarpines.

From the hydrophilic extract of the ascidian Polycarpa aurata three new compounds, N-(4-methoxybenzoyl)-N'-methylguanidine (1), butyl 2-(4-methoxyphenyl)-2-oxoacetate (2), and 2-(4-methoxyphenyl)-N-methyl-2-oxoacetamide (3), together with the known compounds methyl 2-(4-methoxyphenyl)-2-oxoacetate (4) and 4-methoxybenzoic acid were isolated. The structures of all isolates were determined from their spectroscopic data (NMR, MS, IR, UV).