Antifouling activity in some benthic Antarctic invertebrates by “in situ” experiments at Deception Island, Antarctica
Introduction
Some marine organisms, such as algae, sponges, corals and ascidians produce antifouling substances which keep them free from undesirable encrusting organisms in nature (Hentschel et al., 2001, Dobretsov and Qian, 2002, Harder et al., 2003). The settlement of organisms on the surfaces of living organisms (i.e. epibiosis) can be both advantageous and disadvantageous to the host. Advantages may include the production of antifouling substances by symbiotic epibiota (Walls et al., 1993, Harder et al., 2003, Piel, 2004) and furnishing the host with nutrients (Mercado et al., 1998, Faulkner, 2000). Disadvantages may be growth inhibition, necrosis, or death of hosts (Wahl and Mark, 1999). At the beginning of adhesion, bacteria colonize the surface and build up a biofilm (Kirchman and Mitchell, 1981). These microbial biofilms form in a multi-step process of signaling and regulatory events, all of which lead to the development of sessile microbial communities encased in an extracellular polymer matrix. Therefore, microbial biofilms can be disrupted via interference with the signal exchange that leads to biofilm formation or through degradation of the various extracellular polymers that make up the matrices of the biofilms (Dobretsov et al., 2013). Interference with bacterial quorum sensing (QS) has been proposed as a potential approach for controlling biofouling (Dobretsov et al., 2009, Qian et al., 2009, Choudhary and Schmidt-Dannert, 2010, Xiong and Liu, 2010). Quorum-sensing pathways, which allow density-dependent changes in bacterial phenotype, modulate biofilm formation as well as virulence and symbiosis (Davies et al., 1998, Callow and Callow, 2006, Krug, 2006). Although this type of information is generally lacking in marine ecosystems (Clare, 1996), mechanisms by which marine organisms inhibit the settlement of fouling have been investigated at different levels, from molecular to ecological approaches (Steinberg et al., 1998, Golberg et al., 2011, Dobretsov et al., 2013). The extent of microbial colonization on soft-body marine organisms is probably influenced by the chemical effects of bioactive metabolites produced either by the host itself or by symbiotic microorganisms (Lee et al., 2001, Kelly et al., 2003). The ubiquity of fouling organisms in the marine environment and the negative consequences of fouling are likely strong evolutionary pressures for marine organisms to develop defenses to protect their surface from fouling (Wahl, 1989, Steinberg et al., 1997, Maximilien et al., 1998). The planktonic pool of free bacteria, algal spores and competent larvae imposes a strong selective pressure on benthic invertebrates (Krug, 2006). One type of defense against this is the production of secondary metabolites, preventing the initial steps in the bacterial fouling process, such as attachment, growth, and surface spreading (Steinberg et al., 1997).
From an ecological perspective, it is important to test, in situ, the invertebrate extracts against the bacteria that they would naturally encounter in the environment. The present study evaluates the potential antifouling activity of some Antarctic invertebrates by comparing crude extracts against controls. Hydrophilic organic extracts were assayed here at concentrations that were volumetrically equivalent to those found in invertebrate tissues to determine their effects on bacterial attachment. Furthermore, this study uses complementary techniques in order to establish the antifouling activity and to describe the Antarctic bacterial community found in Deception Island. These techniques include genetics and confocal microscopy to measure antifouling activity of twelve Antarctic invertebrates, testing different body parts, when possible. The genes used are large enough to provide distinguishing and statistically valid measurements. In general the comparison of 16S rDNA and 18S rDNA allows differentiation between organisms at the genus level across all major phyla in addition to the classification at multiple levels. Currently, culture-independent methods are considered an important tool in identifying a larger part of the microbial community composition, revealing the existence of a high number of as-yet-uncultured microorganisms (Maugeri et al., 2010). Less than 1% of microorganisms can be cultured and these are not representative of the total phylogenetic diversity (Pham and Kim, 2012). The sequence of the 16S and 18S ribosomal RNA gene is a powerful tool for the taxonomic classification of bacteria and eukaryotes respectively.
Thus, the aims of this study is 1) to validate the methodological approaches to measure the antifouling properties of marine invertebrates extracts, establishing which invertebrates may be of interest for chemical studies from the sponges, cnidarian, bryozoan and tunicate tested and 2) to describe the composition of the bacterial community present in Deception Island waters.
Section snippets
Sample collection and processing
A representative group of Antarctic invertebrate fauna (specimens from four common phyla; porifera, cnidarian, bryozoan and chordate) was selected to evaluate the importance of the antifouling activity in the Antarctic benthos. Eight species of sponges, two cnidarians, one bryozoan and one tunicate were selected for the experiment (Table 1). All these invertebrates were collected in the Eastern Weddell Sea during the ANT XXI/2 cruise of R/V Polarstern (AWI, Bremerhaven, Germany), from November
Bacterial analysis
The differences between the experimental and control treatments were determined by 1-way ANOVA, adopting treatment as a fixed factor. All the six 1-way ANOVA performed (two methacrylates and three response variables) showed a significant p-value (p < 0.05). After 28 days, confocal fluorescence revealed that amounts and densities of bacteria on the gels surfaces were significantly different among some treatments and controls (see Fig. 2, Fig. 3). As both methacrylates were analyzed separately
Discussion
Chemically mediated behavior is widely present in the sea and has considerable impact on the structure and function of marine populations, communities, and ecosystems. Some hydrophilic extracts possess antimicrobial activity reducing bacterial abundance and diversity on their surfaces. Competition for space represents an ecological force comparable to predation and imposes a strong selective pressure on benthic invertebrates. Marine biofouling is the consequence of a wide range of physical,
Conclusion
Even though many natural products can inhibit biofouling in laboratory conditions, very little is known about their performance in the field. From an ecological point of view, it is important to see whether these compounds are produced “in situ” and whether they are produced in quantities that are sufficient to trigger meaningful responses in marine organisms. Thus, here we proved that some invertebrate fractions exhibited antifouling activity measured “in situ” in Antarctic waters. Although in
Acknowledgments
Thank are due to F.J. Cristobo, L. Nuñez-Pons, B. Figuerola, J. Moles, A. Riesgo and J. Vazquez for their support in the lab and in the Antarctic cruise. Thanks are also due to the crew of BAE Gabriel de Castilla for their logistic support during the dives, as well as to M. Postigo and E. García-López for their sequencing support, and Dr. Taboada for the maps. This work was made possible through funding to C. Angulo-Preckler by the FPU Program (AP2009-2081) as well as ACTIQUIM-II (
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