public read Prof. Fabiano Thompson Federal University of Rio de Janeiro Assistent Professor

Av. Carlos Chagas Fo. S/N CCS - IB - Laboratory of Microbiology e SAGE-COPPE - BLOCO A (Anexo) A3 - sl 102 RIo de Janeiro RJ 21941-599 Brazil

+55 21 3938-6567 +55 21 3938-6567 CCS - IB - Laboratory of Microbiology e SAGE-COPPE - BLOCO A (Anexo) A3 - sl 102 http://www.microbiologia.biologia.ufrj.br The health of the coral reefs of the Abrolhos Bank (southwestern Atlantic) was characterized with a holistic approach using measurements of four ecosystem components: (i) inorganic and organic nutrient concentrations, [1] fish biomass, [1] macroalgal and coral cover and (iv) microbial community composition and abundance. The possible benefits of protection from fishing were particularly evaluated by comparing sites with varying levels of protection. Two reefs within the well-enforced no-take area of the National Marine Park of Abrolhos (Parcel dos Abrolhos and California) were compared with two unprotected coastal reefs (Sebastião Gomes and Pedra de Leste) and one legally protected but poorly enforced coastal reef (the “paper park” of Timbebas Reef). The fish biomass was lower and the fleshy macroalgal cover was higher in the unprotected reefs compared with the protected areas. The unprotected and protected reefs had similar seawater chemistry. Lower vibrio CFU counts were observed in the fully protected area of California Reef. Metagenome analysis showed that the unprotected reefs had a higher abundance of archaeal and viral sequences and more bacterial pathogens, while the protected reefs had a higher abundance of genes related to photosynthesis. Similar to other reef systems in the world, there was evidence that reductions in the biomass of herbivorous fishes and the consequent increase in macroalgal cover in the Abrolhos Bank may be affecting microbial diversity and abundance. Through the integration of different types of ecological data, the present study showed that protection from fishing may lead to greater reef health. The data presented herein suggest that protected coral reefs have higher microbial diversity, with the most degraded reef (Sebastião Gomes) showing a marked reduction in microbial species richness. It is concluded that ecological conditions in unprotected reefs may promote the growth and rapid evolution of opportunistic microbial pathogens. 2009-01-27 2010-01-27 Abrolhos reefs -38.625 -36.5 -17.0 -19.0 1414176070782

Water samples were collected by divers within the benthic boundary layer, up to 1 m from the bottom, using two Niskin bottles (10 L each). At least three replicates from Niskin bottles were obtained for each parameter in each of the eight locations. Chlorophyll-a, inorganic nutrients, dissolved organic carbon (DOC), Phosphorus and Nitrogen concentrations and microbial abundance was determined. Chlorophyll a samples were collected using positive pressure filtration of 2 L of water. The filters (Millipore HAWP) were extracted overnight in 90% acetone at 4°C and analyzed by spectrophotometry or fluorimetry. For the inorganic nutrients analyses 1 L of water was frozen and was analyzed at laboratory using the following methods: 1) ammonia by indophenol, 2) nitrite by diazotization, 3) nitrate by reduction in Cd - Cu column followed by diazotization, 4) total nitrogen by digestion with potassium persulfate following nitrate determination, 5) orthophosphate by reaction with ascorbic acid, 6) total phosphorous by acid digestion to phosphate, and 7) silicate by reaction with molybdate. Dissolved organic carbon (DOC), Phosphorus and Nitrogen (shown in Table 1 the sum between the organic and inorganic P and N as Total-P and Total-N, respectively). Microbial abundance was determined from three replicates of seawater per site by flow cytometry with Sybr-green (Life Technologies).

Collected seawater was filtered through Sterivex (0.22 μm) by positive pressure. In total 4 L of seawater were filtered in each Sterivex filter. Microbes collected in Sterivex filters were preserved with SET buffer (20% sucrose, 50 mM EDTA and 0.5 mM Tris-HCl) in liquid nitrogen until DNA extraction procedures at laboratory. Seawater metagenomic DNA extraction was performed following alkaline lysis as previously described 21. High quality DNA extracted from Sterivex filters was sequenced using GS FLX Titanium kit (Roche) a pyrosequencing technology.

Fish and benthic assessments were not performed at California Reef due to logistical limitations. Fish counts (N = 20 per site) were made using a nested stationary visual census technique [33] in the same areas in which the photo-quadrats were taken and at the same depths from which the microbes were collected (see the Study area section). Different size categories of fishes were counted in two different sampling radii, with a size limit for individuals to be included in each count. Each sample began with an identification period of 5 minutes in which all species within a 4 m radius (defined by a tape rule laid immediately before census) were listed. After this period, quantitative data were recorded separately for each species. Individuals,10 cm in total length (TL) were counted in a 2 m radius and recorded in two different size categories:,2 and 2–10 cm. Individuals.10 cm TL were counted in a 4 m radius and recorded in four size categories: 10–20, 20–30, 30–40 and.40 cm. The counts of two species of territorial herbivores (Stegastes fuscus and Stegastes variabilis) were pooled because they are difficult to distinguish underwater. Benthic cover was estimated using photo-quadrats (N = 30 per site) as described previously [11]. A mosaic of 15 high-resolution digital images totaling 0.7 m2 constituted each sample. Quadrats were permanently delimited by fixed metal pins and set at random distances along a 20–50 m axis on the tops of reef pinnacles. Relative coral cover was estimated through the identification of organisms below 300 randomly distributed points per quadrat (i.e., 20 points per photograph) using the Coral Point Count with Excel Extensions software [38]. The counts of benthic organisms were converted to percentages. One-way analysis of variance [34] was used to evaluate differences in benthic cover and fish biomass between the sites. To satisfy the ANOVA assumptions of normality and homoscedasticity, the fish biomass was converted to log (x+1), whereas the benthic cover percentages were converted to arcsin (srt(x)) [35].