ARTHUR DE ALBUQUERQUE TENÓRIO

Shifts between autotrophy and heterotrophy in the reef-building

coral Mussismilia hispida: an approach using fatty acid trophic

markers

Thesis presented at the Oceanographic Institute of the University of São Paulo, as part of the requirements for obtaining the title of Master in Sciences, Oceanography Program, concentration area of Biological Oceanography.

Supervisor: Prof. Dr. Paulo Y. G. Sumida.

São Paulo

2016

ARTHUR DE ALBUQUERQUE TENÓRIO

Shifts between autotrophy and heterotrophy in the reef-building

coral Mussismilia hispida: an approach using fatty acid trophic

markers

São Paulo

2016

Universidade de São Paulo

Instituto Oceanográfico

Shifts between autotrophy and heterotrophy in the reef-building coral Mussismilia

hispida: an approach using fatty acid trophic markers

Arthur de Albuquerque Tenório

Thesis presented at the Oceanographic Institute of the University of São Paulo, as part of the

requirements for obtaining the title of Master of Science, Oceanography Program,

concentration area Biological Oceanography.

Evaluated in: ___/___/____

Prof. Dr. Grade

Prof. Dr. Grade

Prof. Dr. Grade

i

SUMMARY

ACKNOWLEDGEMENTS ................................................................................................................. ii

RESUMO ....................................................................................................................................... iii

ABSTRACT ......................................................................................................................................iv

LIST OF FIGURES ............................................................................................................................ v

LIST OF TABLES ............................................................................................................................. vii

INTRODUCTION ............................................................................................................................. 1

MATERIALS & METHODS ............................................................................................................... 5

Study area and experimental design ......................................................................................... 5

Coral tissue sample processing ................................................................................................. 8

Symbiodinium cell count ........................................................................................................... 8

Fatty acid extraction .................................................................................................................. 9

Gas chromatography ............................................................................................................. 9

Fatty acid trophic markers reference samples ........................................................................ 10

Statistical analyses .................................................................................................................. 12

RESULTS ....................................................................................................................................... 14

Environmental data and Symbiodinium concentration .......................................................... 14

Fatty Acids Trophic Markers reference samples ..................................................................... 21

Fatty Acids Trophic Markers.................................................................................................... 24

Autotrophy vs. heterotrophy – Coral Trophic Index ............................................................... 28

DISCUSSION ................................................................................................................................. 30

LITERATURE CITED ....................................................................................................................... 34

ii

ACKNOWLEDGEMENTS

This work would never been possible to complete without the support of my family. I

will be forever thankful to my Aunt Ana Paula von Sothen and Uncle Otto von Sothen, which

received me in their house and made my stay in São Paulo conceivable. I thanks my parents

Virginia Maria Albuquerque de Araújo and José Ivon Tenório Uchoa for the support, love and

care.

I want to express here my gratitude for the opportunity given to me by my supervisor

Dr. Prof. Paulo Yukio Gomes Sumida. His trust, guidance and friendship were crucial to overcome

all difficulties found during the last two and a half years.

This project would never exist without the help of many friends. I give a special thanks

to PhD student Miguel Mies for the full support, late nights of work and all great time along. Our

lab technician Arthur Z. Güth made my life a lot less miserable, thanks for the support in the

most needed moments. Undergraduation student Jonas Mendes volunteered his time and

strengths during most part of my field trips, without you I would probably still be underwater.

To all other friends from the Instituto Oceanográfico, thanks for the good times, coffee brakes

and everything between.

Friendships completes our lives and makes it more fun. I came to know many amazing

people during this master and I thanks every single one of you that crossed my path. I would like

to thanks Adrian Gonzales and Eric Siciliano for all great times we went through and will go

through. To all friends from Boulder CEPEGE and C.A. da Bio, thank you.

I thanks to CAPES for the scholarship provided. The Oceanographic Institute for the

logistics and operational support and to all employees from Clarimundo de Jesus Research Base

in Ubatuba. It was great to work with all of you.

Lastly, I would like to thanks Clarisse C. L. Miguelote for the best present a person can

receive in life. Thank you for the life of my sun Rafael Miguelote Tenório, and thank you for been

the best mom in the world during my absence.

iii

RESUMO

Os recifes de coral estão entre os ambientes marinhos mais produtivos e ricos em

biodiversidade. Esta biodiversidade está em parte associada a complexas estruturas

formadas por corais escleractíneos. Apesar da importância ecológica, social e econômica

dos recifes de corais, eles são expostos a várias ameaças relacionadas às atividades

humanas. Dentre os impactos antrópicos em recifes, o branqueamento, ou perda de

zooxantelas, é o mais notável e é diretamente relacionado à mortalidade dos corais. Por

possuírem uma associação simbiótica com essas zooxantelas, alguns corais

escleractíneos são considerados mixotróficos, caracterizados por modos de alimentação

autotrófico (através de simbiose com o dinoflagelado Symbiodinium) e heterotrófico

(predação sobre zooplâncton). Alguns estudos comprovam que corais com maior

capacidade de alimentação heterotrófica são mais resistentes ao branqueamento e,

consequentemente, às alterações climáticas. A fim de analisar se o coral escleractíneo

Mussismilia hispida, é capaz de alternar seu modo nutricional entre predominante

autotrófico e predominante heterotróficos, dezoito colônias foram amostradas ao longo

de um ano. Marcadores Tróficos de Ácidos Graxos (FATM, na sigla em inglês) foram

utilizados para determinar a fonte nutricional de carbono em tecido de corais. A

concentração de células de Symbiodinium e a temperatura local também foram

avaliadas. Branqueamento foi observado nos meses mais quentes do ano, quando a

concentração de Symbiodinium diminuiu, voltando a aumentar nos meses mais frios. O

marcador para dieta heterotrófica CGA (C20: 1ω9) foi encontrado em amostras de

zooplâncton de toda a área de estudo. Em laboratório, colônias sem acesso a

zooplâncton apresentaram perda significativa deste marcador após 10 dias. Amostras

de colônias naturalmente branqueadas não apresentaram nenhum vestígio dos

marcadores de autotrofia SDA (18: 4ω3) e DPA (22: 5ω3), mas continham tanto CGA e

DHA (22: 6ω3). Isso confirmou que SDA e DPA são marcadores autotróficos viáveis e

CGA é um marcador de heterotrofia. FATM relacionados com autotrofia apresentaram

padrão semelhante ao observado para as concentrações de Symbiodinium e foram

positivamente correlacionados com a densidade numérica de simbiontes e

negativamente com a temperatura. Para explorar os dados de concentração dos FATM,

o Índice Trófico de Corais foi desenvolvido para exibir as alternâncias entre modos

nutricionais. Mussismilia hispida de fato alterna entre predominância de modo nutritivo

ao longo do ano, sendo mais heterotrófica em períodos mais quentes e em condições

climáticas adversas, porem na maior parte do ano é predominantemente autotrófica. A

validação dos ácidos graxos marcadores tróficos específicos como referência para

autotrofia e heterotrofia em corais abre perspectivas para novos estudos em ecologia

trófica bêntica em recifes de coral. Este trabalho também inclui o primeiro

monitoramento de um ano do comportamento alimentar em um coral hermatípico no

Atlântico Sul e o acompanhamento de um evento de branqueamento.

PALAVRAS-CHAVE: Autotrofia, Branqueamento, Coral, FATM, Heterotrofia, Mixotrofia,

Symbiodinium.

iv

ABSTRACT

Coral reefs are among the most productive and biodiverse marine environments. This

remarkable biodiversity is partly associated to the complex structures formed by

scleractinian corals. Despite the ecological, social and economic importance of coral

reefs, they are constantly exposed to several threats mainly related to human activities.

Climate changes are one of the most notable impacts of human activity related to coral

mortality, mainly due to coral bleaching. Some scleractinian corals are proved to be

mixotrophs, displaying both autotrophic (through Symbiodinium) and heterotrophic

(predation on zooplankton) nutrition modes. Many studies emphasize that corals with

greater capability of heterotrophic feeding are more resilient to bleaching and

consequently to climate change. In order to analyze whether the endemic scleractinian

coral Mussismilia hispida is capable of shifting from predominant autotrophic and

predominant heterotrophic in Ubatuba-SP, 18 colonies were sampled monthly for 12

months. The Fatty Acid Trophic Markers (FATM) approach was used to determine the

source of carbon on coral tissues. Symbiodinium cell density and local seawater

temperature were also assessed. A mild bleaching was observed showing a decrease in

Symbiodinium numerical density during warmer months, but increasing in colder

months. Reference samples validated the relation between all selected FATM and its

corresponding nutritional mode. The heterotrophic feeding marker CGA (C20:1ω9) was

found in zooplankton samples collected throughout the study area. Laboratory starved

colonies (no access to zooplankton) lost any trace of this marker after 10. Samples from

naturally bleached colonies presented no traces of the autotrophic feeding markers SDA

(18:4ω3) and DPA (22:5ω3), but contained both CGA (C20:1ω9) and DHA (22:6ω3).

These results confirmed that the FATM analyzed where reliable trophic markers.

Autotrophic FATM presented a pattern similar to that observed for Symbiodinium

concentration in M. hispida tissues and were positively correlated with the symbiont and

negatively with temperature. The Coral Trophic Index showed that M. hispida undergoes

shifts in nutritional modes along the year, being more heterotrophic in adverse

conditions. The validation of specific FATM as proxies for autotrophic and heterotrophic

feeding in corals opens new perspectives for further studies in benthic trophic ecology

in coral reefs. This work also presents the first yearlong monitoring of the feeding

behavior in a hermatypic coral in the South Atlantic and the monitoring of a mild

bleaching event.

KEYWORDS: Autotrophy, Bleaching, Coral, FATM, Heterotrophy, Mixotrophy,

Symbiodinium.

v

LIST OF FIGURES

Figure 1. Sampling locations of Mussismilia hispida colonies along the coast of Ubatuba.

1: Anchieta I 2: Anchieta II 3: Fortaleza I 4: Fortaleza II 5: Lázaro 6: Praia do

Flamengo. Mussismilia hispida colonies were tagged and tissues were sampled

monthly in each location. Samples were analyzed for Symbiodinium

concentration and fatty acids related to autotrophic and heterotrophic feeding.

Illustration by Romina V. Barbosa. ...................................................................... 6

Figure 2. Mussismilia hispida colonies found in Ubatuba. Three colonies were selected

at each location in similar conditions, including orientation in the substrate,

depth (3 m ± 0.75 m), and absence of fouling and competitors such as frondose

macroalgae. ......................................................................................................... 7

Figure 3. (A) HOBO data logger (Onset®, accuracy of ± 0.53°C; resolution of 0.14°C at

25°C) for the continuous recording of temperature; (B) Structure built to fix

loggers on the substrate next to Mussismilia hispida colonies; (C) Fouling on

data logger and structure after 35 days at sea. .................................................. 7

Figure 4. Symbiodinium cells (arrows) from the coral Mussismilia hispida in the Nageotte

counting chamber. (A) 10 x magnification; (B) 40 x magnification. .................... 9

Figure 5. Experimental set up to assess the decrease of heterotrophic FATM (CGA – cis

gondoic acid) contents in Mussismilia hispida tissue under starving conditions

(i.e., absence of zooplankton). Left: Recirculated aquaria built for keeping the

colonies. Right: Close up of the experimental colonies in the beginning of the

experiment. ....................................................................................................... 11

Figure 6. Bleached Mussismilia hispida colonies sampled in situ to assess the autotrophic

FATM (SDA, DPA and DHA) content and Symbiodinium cells concentration in

the coral tissue. ................................................................................................. 11

Figure 7. Seawater temperature from each Mussismilia hispida coral colony site in

Ubatuba along the sampling period. The horizontal line represents the local

mean temperature. ........................................................................................... 14

Figure 8. Average Symbiodinium cell concentration of Mussismilia hispida samples at

each site through a year cycle (standard deviation not shown for graphic

reasons – see Fig. 9). ......................................................................................... 16

Figure 9. Average Symbiodinium cells concentration in Mussismilia hispida colonies

collected monthly on the six sampling sites at Ubatuba. Vertical lines represent

the standard deviation. ..................................................................................... 17

vi

Figure 10. Average Symbiodinium cell concentration in Mussismilia hispida tissue for all

locations and average water temperature along year cycle in Ubatuba (n=18

colonies). Vertical lines with diamonds are the standard deviation of

Symbiodinium and the light grey lines are the standard deviation of the average

temperature. ..................................................................................................... 18

Figure 11. Symbiodinium cell concentration in Mussismilia hispida for each site and the

average monthly water temperature in Ubatuba. Vertical lines represent the

standard deviation. ........................................................................................... 19

Figure 12. Pearson correlation between Symbiodinium cell concentration from

Mussismilia hispida corals and seawater temperature at Ubatuba. ................ 19

Figure 13. Box Plot for Symbiodinium cell concentrations of bleached Mussismilia hispida

colonies found in Ubatuba (n=3). The horizontal line represents the median and

the diamond, the mean. .................................................................................... 20

Figure 14. Box Plot for Symbiodinium cell concentrations of all Mussismilia hispida

colonies from Ubatuba (n=189). The horizontal line represents the median and

the diamond, the mean. The red X are outliers and vertical lines are lower and

upper whiskers. ................................................................................................. 20

Figure 15. Monthly average concentration of the fatty acid trophic marker cis-gondoic

acid (CGA) for the zooplankton samples collected in the six experimental sites

in Ubatuba. Vertical lines represent the standard deviation. ........................... 21

Figure 16. Starving experiment on Mussismilia hispida colonies. Samples were taken

every two days for 10 days. (A) Docosahexaenoic acid - DHA; (B) Stearidonic

acid - SDA; (C) Docosapentaenoic acid - DPA; and (D) Cis-gondoic acid - CGA.

The gray line indicates the Symbiodinium cell concentration. Vertical lines

represent the standard deviation. .................................................................... 22

Figure 17. Fatty acid composition of bleached Mussismilia hispida colonies (BL 1-3)

performed with naturally bleached found in Ubatuba. The fatty acid C20:1ω9

(CGA) is the heterotrophy FATM while C18:4ω3 (SDA), C22:5ω3 (DPA) and

C22:6ω3 (DHA) are the autotrophy FATM. The value for SDA and DPA is zero for

all samples. DHA value is zero for BL3. ............................................................. 24

Figure 18. Monthly average of Symbiodinium concentration and average concentration

of (A) C18:4ω3 (SDA), (B) C22:5ω3 (DPA) and (C) C20:1ω9 (CGA) of Mussismilia

hispida in Ubatuba. Vertical lines (continuous and dashed) represent the

standard deviation. ........................................................................................... 25

Figure 19. Monthly average temperature and average concentration of (A) C18:4ω3

(SDA), (B) C22:5ω3 (DPA) and (C) C20:1ω9 (CGA), from Mussismilia hispida in

Ubatuba. Vertical lines represent the standard deviation. ............................... 26

vii

Figure 20. Pearson correlation between temperature and SDA (C18:4ω3) concentrations

from Mussismilia hispida corals at Ubatuba. .................................................... 27

Figure 21. Pearson correlation between temperature and DPA (C22:5ω3) concentrations

from Mussismilia hispida corals at Ubatuba. .................................................... 27

Figure 22. Mussismilia hispida Coral Trophic Index (CTI) representing the differences

between the ratios of autotrophy fatty acids trophic markers (SDA, C18:4ω3

and DPA, C22:5ω3) and heterotrophy fatty acid trophic marker (CGA, C20:1ω9).

Positive values indicate a predominance of autotrophic input to the corals’

whereas values below zero indicate the prevalence of heterotrophy. ............ 28

Figure 23. Coral Trophic Index with starved colonies experiment FATM data. The index

is representing the differences between the ratios of autotrophy fatty acids

trophic markers (SDA, C18:4ω3 and DPA, C22:5ω3) and heterotrophy fatty acid

trophic marker (CGA, C20:1ω9). Positive values indicate a predominance of

autotrophic input to the corals’ fatty acids content and values below zero

represent a prevalence of heterotrophy........................................................... 29

LIST OF TABLES

Table 1. Geographic coordinates for each site of Mussismilia hispida sampling. ............ 6

Table 2. Weekly and monthly wave height (m) and direction (WH/D). Weekly, monthly

and between sampling percentage of cloud cover (%CC) and weekly, monthly

and between sampling accumulated rain (AR) for Ubatuba-SP, Brazil (CC and

WH/D source CPETC/INPE, AR source NAP-Oceano Sustentável USP. ............. 15

Table 3. Starving experiment 01 ANOVA and Tukey’s post-hoc test results for the

concentration of Symbiodinium, stearidonic acid (SDA), docosapentaenoic acid

(DPA) and cis-gondoic acid (CGA) in the five (Day2 to Day 10) sampling periods

in the starved colonies. ..................................................................................... 23

1

INTRODUCTION

Coral reefs are among the most productive and biodiverse marine environments

(Connell, 1978; Schnell, 2004; Sheppard et al., 2009). Even though they are mostly found

under oligotrophic conditions, coral reefs are highly productive ecosystems because of

the photosynthesis performed by symbiotic dinoflagellates and macroalgae (Falter et

al., 2011). Despite occupying an area less than 300,000 km2 corresponding to 1% of the

ocean surface area (Sheppard et al., 2009), this ecosystem provides habitat for more

than 25% of the known marine species, including one third of marine fish species, 5,000

mollusk species, 800 hard corals, and a large number of echinoderms, polychaetes,

sponges and crustaceans (UNEP, 2003, Schnell, 2004; Lalli and Parsons, 2006 Sorokin,

2013). With the exception of Onychophora, all metazoan phyla are found in coral reefs

making this the most diverse environment on Earth at higher taxonomic levels (Glynn

and Enochs, 2011).

The high biodiversity of coral reefs is of relevant economical importance, since

approximately 10 million people depend directly on them (Salvat, 1992; Wilkinson,

2008). The main commercial activities related to coral reefs include tourism (diving

centers, hotels and restaurants – see Moberg and Folke, 1999), fisheries (fishes such as

parrotfishes and groupers are important examples of coral reef species harvested for

human consumption – see Bellwood and Wainwright, 2002), production of

pharmaceuticals and medication (Cinel et al., 2000; Alves de Lima et al., 2013) and the

marine aquarium trade (more than 1,000 ornamental fish species are commercially

targeted in an economic activity that is worth more than U$ 15 billion – see Wabnitz et

al., 2003; Olivotto et al., 2006). These activities generate roughly U$ 797 billion every

year (Cesar et al., 2003).

Despite the ecological, social and economic importance of corals reefs, they are

constantly exposed to several threats mainly related to human activities (Roberts et al.,

2002; Burke et al., 2011). These include marine pollution, overfishing, habitat

destruction and climate change associated with carbon dioxide emissions that alter

water temperature and carbonate chemistry (Calado, 2006; Hoegh-Guldberg et al.,

2007). These have destroyed approximately 20% of coral reefs worldwide, while another

2

35% are seriously threatened (Wilkinson, 2008). In some cases, anthropogenic impacts

to coral reefs have exceeded their regenerative capacity, causing dramatic shifts in

species composition and severe biodiversity reduction, which directly led to economic

loss (Bellwood et al., 2004).

The remarkable biodiversity found in coral reefs is partly associated to the complex

calcium carbonate structure formed by scleractinian corals (Cnidaria: Anthozoa). These

are the true reef-building organisms, also called hermatypic, which provide structural

habitat for numerous other species (Krueger et al., 2015). Surface irregularities, crevices

and tunnels in the reef limestone form a variety of microhabitats and the erosion of the

main reef structure creates areas of rubble and sand around it (Hallock, 1997). Most

hermatypic corals live in clear, oligotrophic and shallow tropical waters (Sheppard et al.,

2009). The rate at which hermatypic corals deposit calcium carbonate depends on the

species, but some of the branching species (e.g., Acropora spp.) can grow approximately

10 cm per year (Ruppert et al., 2004), thus making coral reef damage recovery a slow

and fragile process.

Some species of hermatypic corals live in a mutualistic association with unicellular

photosynthetic dinoflagellates of the genus Symbiodinium, also known as zooxanthellae

(Stat et al., 2006; Venn et al., 2008). The coral host provides shelter, nitrogen,

phosphorus and carbon dioxide to the symbiotic dinoflagellate, which in return

translocates up to 95% of its photosynthetically-fixed carbon to the host (Falkowski et

al., 1984; Muscatine and Weis, 2013). Symbiodinium dinoflagellates may contribute up

to 100% of the coral daily metabolic needs (Papina et al., 2003; Grottoli et al., 2006;

Hoegh-Guldberg et al., 2007). For many years reef-building corals were considered

mainly dependent on the photoautotrophy performed by Symbiodinium (Muscatine and

Porter, 1977). However, photosynthetic products may be deficient in nitrogen and

phosphorus (Crossland, 1987; Wild et al., 2004) and essential nutrients for growth and

reproduction may need to be acquired through a different feeding source (Ferrier-Pagès

et al., 2003).

Numerous studies have confirmed that most coral species can in fact be active

heterotrophs and prey on plankton organisms (Sebens et al., 1996; Grottoli, 2002;

Houlbrèque et al., 2004; Palardy et al., 2005, 2006; Houlbrèque and Ferrier-Pagès,

3

2009). Heterotrophic input is necessary for maximal coral growth and it can contribute

up to 66% of the fixed carbon found in both coral tissue and skeleton (Grottoli and

Wellington, 1999; Houlbrèque et al., 2003; Palardy et al., 2005). The ability to feed as

both autotrophs and heterotrophs renders corals the status of mixotrophs.

Mixotrophy supplies corals with different compounds, including a wide range of lipid

compounds (Crossland et al., 1980; Grottoli et al., 2006), which play an essential role in

coral nutrition and metabolism. Among these, fatty acids (FA) are important since they

may be used in ecological studies as trophic markers (i.e., fatty acid trophic makers -

FATM) (Sargent, et al., 1990; Volkman, 1999; Dalsgaard et al., 2003). Animals, differently

from many phototrophs, cannot produce FA with unsaturated bonds beyond a

determinate carbon position (position Δ9; Dewick, 1997). Moreover, animals are

incapable of synthesis de novo for most FA (Dalsgaard et al., 2003) and these are

produced through different cellular pathways. In animals, this occurs in the

phospholipids of the endoplasmic reticulum, whereas in most autotrophs FA are

synthesized in the thylakoid membranes of chloroplasts (Papina et al., 2003). The only

manner in which animals can obtain polyunsaturated fatty acids is by consuming

organisms that produce them (such as phytoplankton) or organisms that have consumed

a producer (Persson and Vrede, 2006). Specific FA obtained in such ways may then serve

as food-source markers (Sargent et al., 1990; Dalsgaard et al., 2003).

Many studies used the approach of FATM to evaluate carbon source in scleractinian

corals (Wilson et al., 2001; Treignier et al., 2008; Dodds et al., 2009; Figueiredo et al.,

2012). It is known that specific FATM in coral tissues may be traced back to the

photosynthetic contribution of Symbiodinium, such as 18:4ω3, 22:5ω3 and 22:6ω3

(Papina et al., 2003; Treignier et al., 2008). Other FA may be specific to heterotrophy,

such as those produced by crustacean zooplankton (e.g., C20:1ω9 and C22:1ω11;

Dalsgaard et al., 2003), which are relevant prey items for hard corals.

However, both autotrophy and heterotrophy may be regulated by abiotic factors.

Increased seawater temperature is associated with declines in photosynthetically-

driven carbon fixation rates (Clark and Jensen, 1982) and with decrease in calcification

rates, tissue biomass and FA reserves (Grottoli et al., 2006; Rodrigues and Grottoli, 2006,

2007; Cantin et al., 2010; Levas et al., 2013). High temperatures and excessive light

4

exposure are the main causes of coral bleaching, which is the paling or whitening of

corals as a result of the loss of Symbiodinium or a decrease in its pigment content

(Douglas, 2003). In addition, ex situ experiments show that corals upregulate

heterotrophic feeding when photosynthesis is suppressed either due to decreased

water clarity and deprivation of sun light (Anthony and Fabricius, 2000; Hoogenboom et

al., 2010) or when symbionts are lost from coral tissue due to bleaching (Grottoli et al.,

2006). The local zooplankton community composition may as well affect heterotrophic

intake because different species of coral prey on certain zooplankton size ranges

(Palardy et al., 2006). However, many studies have demonstrated that not all species

are capable of using heterotrophy to compensate for reduced photosynthesis (Anthony

and Fabricius, 2000; Grottolli et al., 2006). Despite all of this information, no study has

monitored in situ if hermatypic corals shift between feeding modes and what may

regulate these shifts.

The present work aimed to determine if the scleractinian coral Mussismilia hispida

is both autotrophic and heterotrophic and to investigate the influence of abiotic factors

on the relative intensity of those trophic modes. This work is based on the following

hypotheses: i) Mussismilia hispida shifts its feeding mode between predominantly

autotrophic and predominantly heterotrophic; and ii) Mussismilia hispida is

predominantly heterotrophic throughout the year. In order to answer these questions,

we monitored the concentration of specific FATM related to both autotrophy and

heterotrophy in M. hispida colonies for a year at Ubatuba (São Paulo state, Brazil), while

also taking continuous measurements of seawater temperature.

Mussismilia hispida is an endemic and hermatypic species of the Brazilian coast and

Ubatuba is almost at its distribution limit to the south being the only hermatypic coral

species found in the São Paulo state. Understanding how this species feeds and thrives

at such higher latitudes may be important not only for coral reef trophic ecology, but

also may contribute to the current knowledge of possible habitat expansion of more

resistant species and adaptation facing climate change.

5

MATERIALS & METHODS

Study area and experimental design

Sampling took place in the coastal region of the town of Ubatuba (São Paulo

state), Brazil (23°30'01.9"S 45°07'07.9"W). This area is under the influence of the

relatively warm and low salinity Coastal Water (CW) and seasonal summer intrusions of

the South Atlantic Central Water (SACW), with lower temperatures and higher salinity

(Castro Filho et al., 1987). Sea surface temperature in the area ranges from 14.6 to

27.9°C and salinity from 31.7 to 35.8 ppt (Castro, 1995). Low intensity waves come from

the East and stronger waves come from South and Southwest (Castro Filho et al., 1987).

Fine sand, silt and clay predominate, while coarser material is found in enclosed areas

(Mahiques, 1995)

Six experimental sites were selected in Flamengo and Fortaleza Bays and

Anchieta Island (Fig. 1). In each site, three colonies of the Brazilian endemic scleractinian

coral Mussismilia hispida (Fig. 2) were tagged and monthly sampled. In order to have

replicated individuals, colonies were chosen in areas with similar conditions, i.e., similar

position/orientation on the substrate, depth of 3 m ± 0.75 m, direct exposure to sunlight

and near absence of competing organisms such as turf and frondose algae and sessile

invertebrates like barnacles and mussels. All colonies were sampled nearly monthly for

a year (Feb/2015 – Jan/2016), with intervals varying from 20 – 40 days, depending on

climate and hydrodynamic conditions. Approximately one polyp from each colony was

collected monthly with hammer and chisel. Immediately after removal, polyps were

placed in tagged plastic bags with seawater and refrigerated on ice.

Seawater temperature was continuously recorded at each site using HOBO

Onset®data loggers (accuracy of ± 0.53°C; resolution of 0.14°C at 25°C) (Fig. 3). In every

sampling event data loggers were cleaned and the recorded information was retrieved.

All temperature data are presented as a continuous measurement for each site. The

local mean temperature was calculated individually for each site using the whole data

set, specific to each site. Temperature means used for comparisons with Symbiodinium

6

concentration and fatty acid trophic markers (FATM) were calculated using all of the

temperature data recorded in between each sampling period.

Figure 1. Sampling locations of Mussismilia hispida colonies along the coast of Ubatuba. 1: Anchieta I 2: Anchieta II 3:

Fortaleza I 4: Fortaleza II 5: Lázaro 6: Praia do Flamengo. Mussismilia hispida colonies were tagged and tissues were

sampled monthly in each location. Samples were analyzed for Symbiodinium concentration and fatty acids related to

autotrophic and heterotrophic feeding. Illustration by Romina V. Barbosa.

Table 1. Geographic coordinates for each site of Mussismilia hispida sampling.

Location Coordinates

1 Anchieta I S 23°32'02.5404"; W 45°04'40.8144"

2 Anchieta II S 23°32'39.2820"; W 45°04'46.3800"

3 Fortaleza I S 23°31'52.2372"; W 45°09'44.0748"

4 Fortaleza II S 23°31'46.4232"; W 45°09'31.4064"

5 Lázaro S 23°30'30.2364"; W 45°08'11.0904"

6 Praia do Flamengo S 23°30'37.6164"; W 45°06'29.5200"

7

Ocean and climate data were also obtained from CPTEC/INPE (Centro De

Previsão de Tempo e Estudos Climáticos/Instituto Nacional de Pesquisas Espaciais) and

monthly-accumulated rainfall was obtained from NAP-Oceano Sustentável (Núcleo de

apoio à pesquisa oceano sustentável). Percentage of cloud coverage data and mean

wave height and direction were compiled for each week and month of 2015 and for

January 2016. For the periods in between samplings, accumulated rain and percentage

cloud coverage were calculated as well.

Sampling was not performed in July due to rough weather, as waves were

frequently higher than 2.0 m and hindering sampling conditions. Tagged colonies from

Anchieta II were not sampled in July, September and October, as they were not found

due to poor water visibility and weather conditions.

Figure 2. Mussismilia hispida colonies found in Ubatuba. Three colonies were selected at each location in similar conditions, including orientation in the substrate, depth (3 m ± 0.75 m), and absence of fouling and competitors such as frondose macroalgae.

Figure 3. (A) HOBO data logger (Onset®, accuracy of ± 0.53°C; resolution of 0.14°C at 25°C) for the continuous recording of temperature; (B) Structure built to fix loggers on the substrate next to Mussismilia hispida colonies; (C) Fouling on data logger and structure after 35 days at sea.

8

Coral tissue sample processing

Major skeleton fragments were removed with pliers and tweezers. Each sample was

then divided in two aliquots of approximately 0.1 g each. The aliquots were used for (i)

Symbiodinium cell count and (ii) fatty acid extraction for gas chromatography analysis.

Symbiodinium cell count

Skeleton fragments were removed from soft tissue through sample homogenization

and decantation. Excess water from the tissues was removed by briefly stirring the

sample on a dry petri dish followed by weighing. Samples were then transferred to 2-mL

centrifuge tubes with 1 mL of distilled water. The contents were grinded with a pestle,

homogenized for 5 minutes and agitated for 3 minutes. The resulting solution was

filtered through a 100-µm mesh in order to remove unwanted debris, and then washed

with 4 ml of distilled water. The final filtered solution had a volume of 5 ml.

Symbiodinium cells were counted in a Nageotte counting chamber (Fig. 4). The number

of Symbiodinium cells per mg of coral soft tissue was obtained according to equation (1).

(1) 𝑆𝑦𝑚𝑏/𝑚𝑔𝑐𝑠𝑡 =(1,25𝐶𝐶×1000𝑁𝐿)×5

𝑆𝑊

Where:

𝑐𝑠𝑡 - Coral soft tissue;

CC - Counted Symbiodinium cells;

NL - Counted Nageotte chamber lines;

SW - Coral soft tissue weight.

9

Figure 4. Symbiodinium cells (arrows) from the coral Mussismilia hispida in the Nageotte counting chamber. (A) 10 x magnification; (B) 40 x magnification.

Fatty acid extraction

Lipids were extracted from samples using a method adapted from Bligh & Dyer

(1959). The soft tissue (skeleton-free) was weighed and added to a glass tube. One

milliliter of a solution of 100% methanol and 100% dichloromethane (1 : 1) was added

to each tube and agitated for one minute, followed by the addition of 100 µL of internal

standard (tricosanoic acid, C23:0). Samples were then esterified with 5 mL of a 1 : 0.02

solution of methanol and sulfuric acid. Subsequently, 2 mL of 100% hexane was added

and agitated. Condensers were attached to the tubes and placed in a 50°C bath for 30

minutes. The tubes were cooled to room temperature and phases separated with a 5

mL saturated solution of NaCl. The top phase was retrieved (2 mL) for GC analysis. When

samples weighed less than 50 mg, concentration to 1 mL was performed by evaporation

with pure (99.999%) Nitrogen gas.

Gas chromatography

Esterified samples were analyzed in a 6890 Agilent gas chromatograph coupled

with a flame ionization detector (GC-FID). The column used was the Agilent J & W (50 m

long, 0.35 mm internal diameter and 0.17 mm thick with a 5% phenylmethylsiloxane

film). Two microliters of the sample were injected and carried by ultra-pure hydrogen

and nitrogen gases. The oven ramp temperature started at 50 °C, and kept constant for

10

2 minutes, then it increased at the rate of 9°C min- 1, remaining constant until it reached

300°C and maintained for 10 minutes.

The concentrations of four fatty acids were investigated: stearidonic acid (SDA,

18:4ω3), docosapentaenoic acid (DPA, 22:5ω3), docosahexaenoic acid (DHA, 22:6ω3)

and cis-gondoic acid (CGA, C20:1ω9). The first three are markers for autotrophy

(translocated from Symbiodinium to the host – see Papina et al., 2003) whereas the last

one is for heterotrophy (CGA is found in copepods – see Dalsgaard et al., 2003). The

identification of methyl esters of fatty acids was done by comparison of the retention

times of the target fatty acids with a reference analytical standard (47033 PUFA No. 1

Marine Source - Sigma-Aldrich Co.®), based on the calibration curves fitted with at least

5 different concentrations.

Fatty acid trophic markers reference samples

Three different reference samples were produced in order to confirm if the

investigated FATM were adequate. The first group of reference samples was the

monthly night-time collection of zooplankton on each site to confirm the presence of

CGA in zooplankton organisms. This is a marker specific to crustaceans (Dalsgaard et al.,

2003) and was selected as a proxy for heterotrophic feeding. Zooplankton oblique tows

were made with a 300-µm mesh conical net for 5 minutes at 3 knots.

The second group of reference samples was produced through an experiment

that intended to verify if CGA found in the host tissue was indeed related to

heterotrophic feeding. For that purpose, four Mussismilia hispida colonies (Fig. 5) were

maintained for 10 days in a tank with filtered seawater and no zooplankton at a

temperature of 25°C ± 1.8, salinity of 34 and with indirect sunlight. Approximately 70%

of the water was changed daily and samples were taken every two days to monitor the

concentration of CGA in the host tissue and to observe the concentration of

Symbiodinium cells concentration. From hereafter we call these starving colonies. Day

0 were sampled prior to colonies removal from the environment and it was considered

11

as a stress period due to removal disturbances and adaptation to the experiment

system.

The last analysis had the purpose of verifying if the three autotrophy FATM, i.e.,

SDA, DPA and DHA, were specific to Symbiodinium as previously reported (Papina et al.,

2003). For that, we collected bleached colonies in which there would be little or no

Symbiodinium (Fig. 6) and most likely a very small or undetectable amount of these

FATM.

Figure 5. Experimental set up to assess the decrease of heterotrophic FATM (CGA – cis gondoic acid) contents in Mussismilia hispida tissue under starving conditions (i.e., absence of zooplankton). Left: Recirculated aquaria built for keeping the colonies. Right: Close up of the experimental colonies in the beginning of the experiment.

Figure 6. Bleached Mussismilia hispida colonies sampled in situ to assess the autotrophic FATM (SDA, DPA and DHA) content and Symbiodinium cells concentration in the coral tissue.

12

Statistical analyses

Mean temperature differences among sampled months were tested using a one-

way Analysis of Variance (ANOVA), using sites as replicates (one temperature sensor per

site, n=6). Mean temperature differences among sites was tested also with a one-way

ANOVA, using sampled months as replicates (n=11; in October 2015 at Lázaro, the

sensor was lost).

For the starving experiment, each FATM (DHA, SDA, DPA and CGA) was tested

for differences among sampling times using a one-way ANOVA. Differences between

individual sampling times for each FATM were tested for significance with a pairwise

post-hoc Tukey test.

Symbiodinium cell concentrations were tested for differences among sites and

sampling periods using a 2-way nested ANOVA, with the sampled months nested within

each site. If significant difference was found, each pair was compared for significant

differences with a post-hoc Tukey test.

Significant relationships between pairs of variables, such as Symbiodinium

concentration and mean temperature, FATM and mean temperature, and between the

specific FATM (DHA, SDA, DPA and CGA) to Symbiodinium concentration was assessed

using Pearson’s correlation test.

Statistical analyses were performed using the statistical packages JMP (version

8). Biological data (Symbiodinium cell concentration and FATM concentrations) were

log-transformed [log(x+1)] to guarantee homoscedasticity and normalization

requirements for parametric tests (ANOVA and Tukey). All the figures were produced

with Grapher 8 and Microsoft Excel software.

13

Coral Trophic Index

In order to answer the proposed hypotheses, three equations (2-4) were created

using the results of the concentrations of fatty acids for both autotrophy and

heterotrophy. Equations (2) and (3) determine if the intensity of each trophic mode

increased or decreased from one sampling month (t0) to another (t1). The difference

between equations (2) and (3) was calculated to determine which of the trophic modes

predominates in a colony at a specific moment. The resulting value was denominated

Coral Trophic Index (CTI). A positive value reflects a predominantly autotrophic feeding

behavior, whereas negative values indicate a predominantly heterotrophic behavior.

(2) heteroFATMRatio = [(𝑋𝑡1ℎ𝑒𝑡𝑒𝑟𝑜𝐹𝐴𝑇𝑀

𝑋𝑡0ℎ𝑒𝑡𝑒𝑟𝑜𝐹𝐴𝑇𝑀) − 1]

(3) autoFATMRatio = [(𝑋𝑡1𝑎𝑢𝑡𝑜𝐹𝐴𝑇𝑀

𝑋𝑡0𝑎𝑢𝑡𝑜𝐹𝐴𝑇𝑀) − 1]

(4) CTI = (autoFATMRatio − heteroFATMRatio)

Where:

Xt1heteroFATM = [µg g-1] of CGA of sample x at t1

Xt0heteroFATM = [µg g-1] of CGA of sample x at t0

Xt1autoFATM = [µg g-1] of autoFATM (SDA + DPA) of sample x at t1

Xt0autoFATM = [µg g-1] of autoFATM (SDA + DPA) of sample x at t0

14

RESULTS

Environmental data and Symbiodinium concentration

There were no differences in seawater temperature between sites, but there are

significant differences between months of samples (F(10,54) = 41.699, p = 0.001) (Fig. 7).

The lowest temperature was registered in July 2015 at Lázaro (18.6°C), and the highest

in January 2015 at Praia do Flamengo (33.7°C), with an overall year average of 25.5°C.

Figure 7. Seawater temperature from each Mussismilia hispida coral colony site in Ubatuba along the sampling period. The horizontal line represents the local mean temperature.

15

During 2015 a few unusual climatic events were observed. According to the

National Oceanic and Atmospheric Administration (NOAA), one of the biggest El Niño

formations was registered in 2015 in the Pacific Ocean. July/2015 was the warmest

month ever recorded for the globe and Brazil experienced the worst drought in 80 years.

All this reflected in the water conditions for the coast of Brazil. During summer, calm,

warm and clear waters were observed in Ubatuba. In March and April, heavy rains,

winds and rough water were expected, but this was not observed during the

experiment. The water maintained good conditions (calm waters with good visibility) all

the way to June, and rough weather started to hit the Ubatuba coast only after the

second half of June 2015 (Table 2). From the end of June to the beginning of August,

consecutive storms in the coast generated waves higher than two meters. Furthermore,

heavy cloud coverage of 68.3% in October and 82% in November marked the beginning

of the summer 2015/2016 (Table 2). In Brazil, the summer months are typically hot and

humid while winter months are cold and dry.

Table 2. Weekly and monthly wave height (m) and direction (WH/D). Weekly, monthly and between sampling percentage of cloud cover (%CC) and weekly, monthly and between sampling accumulated rain (AR) for Ubatuba-SP, Brazil (CC and WH/D source CPETC/INPE, AR source NAP-Oceano Sustentável USP.

%CC WH/D %CC WH/D %CC WH/D %CC WH/D %CC WH AR (mm) %CC AR (mm)

January/2015 74.6 1.0/S 67.3 1.1/E 71.7 0.9/SE 86.4 1/E 75.0 1.0 246.9 - -

February/2015 55.7 1.2/SSE 44.0 1/ESE 78.3 1.3/SE 53.9 1/E 57.5 1.1 345.6 67.2 512.5

March/2015 40.0 1.4/SSE 73.0 1/E 80.0 1/ESE 53.0 1.3/SSE 62.2 1.2 327 65.0 244.7

April/2015 37.4 1.2/SE 39.0 1/S 66.3 1.1/SSE 30.5 1.5/S 43.6 1.2 180.1 51.0 289.0

May/2015 43.0 1.4/SE 65.5 1.7/S 48.7 1.5/ESE 48.2 1.2/S 49.7 1.4 86.3 46.9 157.2

June/2015 25.8 1.0/E 19.5 1.4/S 34.0 1.7/S 40.0 1.6/E 27.2 1.4 214.6 35.6 21.3

July/2015 97.9 2.1/SSE 55.0 1.1/ESE 55.8 1.5/E 98.0 2.0/S 51.0 1.5 50.7 - -

August/2015 0.8 1.2/SE 5.0 2/E 19.4 1.6/ESE 37.4 1.5/SE 17.0 1.5 16.6 50.1 287.7

September/2015 59.2 1.5/S 73.3 1.3/E 7.7 1.4/SE 47.5 1.2/S 46.4 1.3 169.3 31.3 183.3

October/2015 59.1 1.3/S 62.3 1.4/ESE 73.3 1.4/SE 79.6 1.5/SE 68.3 1.4 217.6 64.4 159.6

November/2015 86.6 1.6/S 77.0 1.0/E 77.0 1.3/SE 86.2 1.3/SE 82.0 1.3 341.2 78.7 299.5

December/2015 90.6 1.3/S 72.2 1.0/S 66.7 1.1/SE 78.8 1.1/E 78.8 1.1 155.8 81.5 303.7

January/2016 89.4 1.5/SE 93.2 1.2/S 64.8 1.1/SE 73.4 1.1/SE 80.2 1.2 278.7 80.1 234.8

Months

Week AverageMonthly

Between

Sampling1 2 3 4

16

Symbiodinium cell concentrations in M. hispida tissue were significantly different

among sites (F(5,53) = 2.541, p = 0.0334), but with a similar variation over time (F(5,53) =

0.780, p = 0.8369) (Fig. 8). In the beginning of the experiment in February, all coral

colonies had a relatively low concentration of Symbiodinium. Overall, throughout the

year the major difference between sites was the absolute numbers while the variation

pattern of Symbiodinium cell concentration was similar for all sites.

Figure 8. Average Symbiodinium cell concentration of Mussismilia hispida samples at each site through a year cycle (standard deviation not shown for graphic reasons – see Fig. 9).

A high variability in Symbiodinium concentration was observed throughout the

experiment (Fig. 9). The average concentration was 8,244.4 ± 5,307.3 cells per mg-1 of

coral soft tissue. Maximum cell concentration was measured at Praia do Flamengo in

April (23,987.9 ± 1,636.5) and Fortaleza II in September (23,202.7 ± 2,072.0) (Fig. 9).

Minimum concentrations were found at Lázaro in February (157.4 ± 55.1) and Fortaleza

I in May (178.4 ± 43.0) (Fig. 9). Anchieta II was the location with higher concentration of

symbionts for the most part of the experiment.

0

5000

10000

15000

20000

[Cel

ls m

g-1]

Anchieta I Anchieta II Fortaleza I

Fortaleza II Lázaro P. Flamengo

17

Figure 9. Average Symbiodinium cells concentration in Mussismilia hispida colonies collected monthly on the six sampling sites at Ubatuba. Vertical lines represent the standard deviation.

Symbiodinium concentration decreased in periods of high seawater

temperatures (29-33°C) that occurred in summer months (November to April) (Figs. 10

and 11). During warmer months, there was a mild bleaching in M. hispida, as colonies

were found with a low Symbiodinium concentration (minimum average 4,563.0 ±

2,437.5), nearly three times less than the maximum average cell concentration 12,946.0

18

± 4132.6 (Fig.10 and 14). However, there were no clear visual signs of bleaching. Visually

confirmed bleached colonies (collected for the third group of reference samples) had an

average of 66.4 ± 9.2 Symbiodinium cells mg-1, which differed to the minimum (157.4 ±

55.1) by nearly two-and-a-half times (Fig. 13 and 14). The opposite trend was observed

in colder months (May to September), where temperature decreases were associated

with a rise in Symbiodinium concentration, as colonies slowly recovered (23,202.7 ±

2,072.0 cells mg-1 in September) (Fig. 10, 11, 13 and 14). An oscillation of 99.3% was

found between maximum and minimum symbiont concentration (Fig. 13 and 14).

Statistically, there was a significant negative correlation between temperature and

Symbiodinium concentration (r(198) = -0.318, p = 0.001 – Fig. 12).

The pattern of high symbiont concentration in winter months vs. low

concentration in summer months is clearer on Anchieta I, Anchieta II and Fortaleza II

(Fig. 11). Symbiodinium concentration observed from March to May in fig. 9 is an

exception to the pattern observed throughout the year. It is clear that an increase in

symbiont concentration in April followed by a decrease in May is found only at Anchieta

I and Praia do Flamengo (Fig. 11), and at this last site it is noticeable a sudden drop in

water temperature (below 20°C). A rapid increase in Symbiodinium concentration in

March was observed at Fortaleza I and Lázaro, but in April the symbiont concentrations

seemed to resume a steady increase through the winter.

Figure 10. Average Symbiodinium cell concentration in Mussismilia hispida tissue for all locations and average water temperature along year cycle in Ubatuba (n=18 colonies). Vertical lines with diamonds are the standard deviation of Symbiodinium and the light grey lines are the standard deviation of the average temperature.

15

20

25

30

0

5000

10000

15000

°C

[Cel

ls m

g-1]

Symbiodinium Average Temp.

19

Figure 11. Symbiodinium cell concentration in Mussismilia hispida for each site and the average monthly water temperature in Ubatuba. Vertical lines represent the standard deviation.

Figure 12. Pearson correlation between Symbiodinium cell concentration from Mussismilia hispida corals and seawater temperature at Ubatuba.

20

Figure 13. Box Plot for Symbiodinium cell concentrations of bleached Mussismilia hispida colonies found in Ubatuba (n=3). The horizontal line represents the median and the diamond, the mean.

Figure 14. Box Plot for Symbiodinium cell concentrations of all Mussismilia hispida colonies from Ubatuba (n=189). The horizontal line represents the median and the diamond, the mean. The red X are outliers and vertical lines are lower and upper whiskers.

21

Fatty Acids Trophic Markers reference samples

All zooplankton samples contained C20:1ω9 (CGA – cis-gondoic acid), averaging

0.3% of the total FA composition. Mean CGA total content was 4.99 ± 3.11 µg g-1 (Fig.

15). Higher CGA concentration was found during winter months and January 2016.

The experiment for the second group of reference samples monitored the

changes in FATM on Mussismilia hispida colonies in the absence of zooplankton

throughout ten days. The FATM profile for the four fatty acids and Symbiodinium cell

concentrations through the whole experiment period is shown in Fig. 16. After the stress

period (Day 0), Symbiodinium concentration stabilizes and there is no significant

difference in its concentration from day 2 to day 10 (F(4,10) = 0.034, p = 0.0526) (Table 3).

All three symbiont-specific fatty acids (18:4ω3 - SDA, 22:5ω3 - DPA and 22:6ω3 – DHA)

presented similar trends to the Symbiodinium concentration and there were no

significant differences on their oscillation (Fig. 16 and Table 3). However, the

concentration of the heterotrophy-specific fatty acid CGA was significantly different

(F(4,9) = 0.042, p = 0.033) (Table 3). The concentration of the heterotrophy specific marker

continuously dropped from D0 to D10 (Fig. 16 D).

0

5

10

15

20

[µg

g -1

]

Figure 15. Monthly average concentration of the fatty acid trophic marker cis-gondoic acid (CGA) for the zooplankton samples collected in the six experimental sites in Ubatuba. Vertical lines represent the standard deviation.

22

Figure 16. Starving experiment on Mussismilia hispida colonies. Samples were taken every two days for 10 days. (A) Docosahexaenoic acid - DHA; (B) Stearidonic acid - SDA; (C) Docosapentaenoic acid - DPA; and (D) Cis-gondoic acid - CGA. The gray line indicates the Symbiodinium cell concentration. Vertical lines represent the standard deviation.

23

Table 3. Starving experiment 01 ANOVA and Tukey’s post-hoc test results for the concentration of Symbiodinium, stearidonic acid (SDA), docosapentaenoic acid (DPA) and cis-gondoic acid (CGA) in the five (Day2 to Day 10) sampling periods in the starved colonies.

Test One-way ANOVA

Tukey’s post-hoc DF F p

Symbiodinium

Sample Time 4 3.410 0.052 D2 D4 D6 D10 D8

Error 10

SDA

Sample Time 4 0.411 0.769 D10 D4 D8 D2 D6

Error 9

DPA

Sample Time 4 0.505 0.733 D2 D4 D10 D8 D6

Error 9 CGA

Sample Time 4 4.236 0.033 D4 D2 D6 D8 D10

Error 9

24

For the last reference samples, the three bleached colonies had an average of

66.4 ± 9.2 Symbiodinium cells, which is more than two orders of magnitude than the

average (7,834.5 ± 5,447.2) found in the Ubatuba area (Figs. 13, 14 and 17). The FATM

composition of the bleached colonies was similar and none of them had detectable SDA

or DPA (Fig. 17). The DHA, however, was found in two of the colonies. From all the FATM,

CGA was the only FA found in all colonies and the most abundant.

Figure 17. Fatty acid composition of bleached Mussismilia hispida colonies (BL 1-3) performed with naturally bleached found in Ubatuba. The fatty acid C20:1ω9 (CGA) is the heterotrophy FATM while C18:4ω3 (SDA), C22:5ω3 (DPA) and C22:6ω3 (DHA) are the autotrophy FATM. The value for SDA and DPA is zero for all samples. DHA value is zero for BL3.

Fatty Acids Trophic Markers

The three markers for autotrophy varied similarly to the Symbiodinium

concentration (SDA - r(198) = 0.375, p = 0.0001; DHA - r(198) = 0.237, p = 0.0008; and DPA -

r(198) = 0.141, p = 0.0473). Their concentrations were low in the beginning of the year

and increased until May, despite the drop in Symbiodinium concentration in this month.

From May to August, the autotrophy FATM decreased, rising again from September

2015 to January 2016 (Fig. 18 A-C).

The concentration of CGA appeared to increase while the other FATM decrease.

The highest concentration of this FATM was found in December 2015 and January 2016

(Fig. 18 D), when symbiont concentration was decreasing. Overall, it had no significant

correlation with Symbiodinium concentration (r(198) = 0.065, p = 0.362).

6,190

3,353 3,1282,578 2,567

00 0 00 0 00

2

4

6

8

10

BL1 BL2 BL3

[µg

g-1]

CGA DHA SDA DPA

25

To better represent the autotrophic feeding behavior, combinations of the three

different markers were tested, and the stronger correlation with Symbiodinium was

found when SDA and DPA were pooled together (r(186) = 0.387, p = 0.001), discarding the

DHA (Fig. 20). The combination of SDA and DPA is from hereafter denominated as

autoFATM.

Figure 18. Monthly average of Symbiodinium concentration and average concentration of (A) C18:4ω3 (SDA), (B) C22:5ω3 (DPA) and (C) C20:1ω9 (CGA) of Mussismilia hispida in Ubatuba. Vertical lines (continuous and dashed) represent the standard deviation.

26

A significantly negative correlation was found between temperature and

autoFATM (SDA r(183) = -0.385, p = 0.0001, and DPA r(183) = -0.154, p = 0.0299) (Fig. 19-

21). CGA did not correlate with temperature (r(183) = -0.032, p = 0.649). A trend in the

concentration of the autoFATM markers with the variation of temperature was noticed.

CGA concentration oscillated differently, reaching higher concentrations in the

beginning of summer (Fig. 19 C).

Figure 19. Monthly average temperature and average concentration of (A) C18:4ω3 (SDA), (B) C22:5ω3 (DPA) and (C) C20:1ω9 (CGA), from Mussismilia hispida in Ubatuba. Vertical lines represent the standard deviation.

27

Figure 20. Pearson correlation between temperature and SDA (C18:4ω3) concentrations from Mussismilia hispida corals at Ubatuba.

Figure 21. Pearson correlation between temperature and DPA (C22:5ω3) concentrations from Mussismilia hispida corals at Ubatuba.

28

Autotrophy vs. heterotrophy – Coral Trophic Index

Ratios for the variation (from one time point to another) of both autotrophy and

heterotrophy FATMs (equations 2 and 3) were produced. Twelve months of sampling

only generated 10 comparison intervals (not 11 because of the missing data in July 2015)

on the coral trophic index. Mussismilia hispida corals were predominantly dependent

on autotrophy during most part of the experiment (6 out of 10 times) (Fig. 22).

Heterotrophy only appeared as the coral main nutrition source in 4 moments, May/Jun,

Ago/Sep, Nov/Dec and Dec/Jan (Fig. 22). There was a predominance of autotrophy in

Feb/Mar, which followed the trend observed for Symbiodinium concentration (Figs. 10-

11). In Apr/May and May/Jun values were close to zero, indicating a balance of both

nutrition modes.

Figure 22. Mussismilia hispida Coral Trophic Index (CTI) representing the differences between the ratios of autotrophy fatty acids trophic markers (SDA, C18:4ω3 and DPA, C22:5ω3) and heterotrophy fatty acid trophic marker (CGA, C20:1ω9). Positive values indicate a predominance of autotrophic input to the corals’ whereas values below zero indicate the prevalence of heterotrophy.

To further explore and validate the CTI, FATM data from the starving experiment

(Fig. 16) was applied to the index and it is displayed on Figure 23. As described for the

experiment, the corals started with a slight predominance of heterotrophy on

Day0/Day2. The deprivation of zooplankton reflected on a loss of CGA and consequently

-1,5

-1

-0,5

0

0,5

1

1,5

CTI

29

on the increase of autotrophy prevalence. By the end of the test, autotrophy increased

by 62% in relation to heterotrophy.

Figure 23. Coral Trophic Index with starved colonies experiment FATM data. The index is representing the differences between the ratios of autotrophy fatty acids trophic markers (SDA, C18:4ω3 and DPA, C22:5ω3) and heterotrophy fatty acid trophic marker (CGA, C20:1ω9). Positive values indicate a predominance of autotrophic input to the corals’ fatty acids content and values below zero represent a prevalence of heterotrophy.

30

DISCUSSION

The present work aimed to determine whether the Brazilian endemic

scleractinian coral Mussismilia hispida is capable of shifting between nutritional modes

and to investigate if heterotrophy is the most predominant mode throughout the year.

To answer the proposed hypotheses, specific fatty acid trophic markers (FATM) were

used to identify the source of fatty acids (FA) present in M. hispida tissues at Ubatuba.

The FATM approach is based on the premise that the selected markers cannot be

selectively processed through food uptake or incorporation and are not synthetized by

the targeted animal, instead they should be transferred from one trophic level to the

next by incorporation or ingestion from the animal nutritional source (Napolitano et al.,

1997; Dalsgaard et al., 2003; Iverson et al., 2004). Despite strong evidence that SDA, DPA

and DHA are translocated from Symbiodinium to its coral host (Papina et al., 2003) and

that CGA is a specific marker for crustacean zooplankton (Dalsgaard et al., 2003), we

produced three reference samples in order to confirm that these markers were

adequate indicators of autotrophic and heterotrophic carbon input for M. hispida.

Scleractinian corals of many different species have been observed to prey on

zooplankton (Ferrier-Pagès et al., 2003; Sebens et al., 2003; Palardy et al., 2005). There

is evidence that within the zooplankton, copepods are the dominant food source for

corals (Dodds et al., 2009) and CGA has been reported as a FATM produced by calanoid

copepods (Dalsgaard et al., 2003). These are the most abundant organisms found in

zooplankton communities at Ubatuba (Lopes, 2003). The first group of reference

samples demonstrated that CGA was found in all zooplankton samples collected at all

locations throughout the year, thus showing that this marker is representative of the

local community.

Under controlled laboratory conditions, M. hispida colonies were placed in

closed and recirculated aquaria without the presence of zooplankton (i.e., in starving

conditions), but with free access to sunlight. The concentration of CGA significantly

decreased during this period and the FATM for autotrophy (SDA, DPA and DHA) did not;

in fact, after the sixth day they increased. Therefore, coupled with the zooplankton

31

samples, these results confirm that CGA can be regarded as an effective trophic marker

for heterotrophic feeding in M. hispida, reassuring the findings in Dalsgaard et al. (2003).

The last reference samples consisted of three highly-bleached colonies that were

found and collected at Ubatuba. Both FATM SDA and DPA were absent and the

heterotrophy marker (CGA) was found at much higher concentrations. This confirms

that that SDA and DPA found in M. hispida are specific to Symbiodinium. The corals were

likely compensating symbiont loss with prey capture (Grottoli et al., 2006). However,

DHA was found in these bleached colonies. This is most likely related to the fact that

DHA is not specific to dinoflagellates, but is also abundantly produced by many

phytoplankton species (Volkman, 1999), and may have been present in zooplankton that

had consumed diatoms and microalgae. Therefore, despite being produced by

Symbiodinium, DHA is an ambiguous marker and was excluded from most of the

analyses. Thus, the combination of SDA and DPA (defined as the autoFATM) is an

adequate FATM for the autotrophic contribution from Symbiodinium to the coral host.

It is important to note that the concentration of SDA and DPA is not a proxy for

the concentration of Symbiodinium in the coral tissue. Production rates of these fatty

acids vary considerably (Titlyanov et al., 2001; Zhukova and Titlyanov, 2002), especially

due to cell size, shape, strain and associated host (Bishop and Kenrick, 1980; Titlyanov

et al., 2001). For instance, Symbiodinium undergoing photoacclimation typically produce

higher concentrations of photosynthates (Anthony and Hoegh-Guldberg, 2003a, 2003b;

Hoogenboom et al., 2006). Furthermore, a very weak correlation was found between

Symbiodinium concentration and the combined content of SDA and DPA (r(186) = 0.387).

Therefore, we consider an increase in SDA and DPA as an increase in their production by

the Symbiodinium population inside the coral tissue.

Our first hypothesis was confirmed as the Coral Trophic Index (CTI) constantly

oscillated between positive and negative values, showing that indeed there are shifts

between predominantly autotrophic and predominantly heterotrophic feeding modes

(Fig. 22). It is generally accepted that most of these shifts are related to turbid waters

(Anthony, 2000; Fabricius and Dommisse, 2000), temperature changes, reduced

photosynthesis and Symbiodinium concentration, as a compensatory strategy (Ferrier-

Pagès et al., 2003; Palardy et al., 2005; Grottoli et al., 2006; Houlbréque and Ferrier-

32

Pagés, 2009). While we expected that temperature would be the major factor driving

theses shifts, as reported in (Rodrigues and Grottoli, 2007; Palardy et al., 2007; Connolly

et al., 2012; Ezzat et al., 2016), we found no correlation between the CTI and

temperature or temperature variations. However, these shifts do coincide with the

sporadic adverse weather conditions reported. These conditions include strong rainfall

from February to March, turbulent hydrodynamics and sediment resuspension from July

to August and increased temperature and cloud cover from October to November (Table

2 and Fig. 7). All these events coincided with shifts from autotrophy to heterotrophy

(Fig. 22). Intense weather has been linked with coral damaging (Crabbe et al., 2008),

while increased rainfall, severe terrestrial runoff and high wave energy have been

reported to cause severe damage, bleaching and/or mortality of coral colonies (Connell,

1997; Connell et al., 1997; Mallela and Crabbe, 2009).

Our second hypothesis was rejected, as the CTI indicates that M. hispida was

predominantly autotrophic, for 6 periods out of 10, which represent the greater part of

the experiment. Heterotrophy was expected to predominate due to the environmental

conditions found at Ubatuba, which is near the limit of the species distribution (e.g., low

seawater temperature in the winter and autumn and high turbidity throughout the

year). These are the main factors that lead the corals to upregulate heterotrophy and

suppress photosynthesis (Anthony and Fabricius, 2000; Grottolli et al., 2006;

Hoogenboom et al., 2010; Levas et al., 2013). Our results show that this species resorts

to heterotrophy when adverse climate conditions seem to hinder photosynthetic

production by Symbiodinium.

Interestingly, our data has shown that M. hispida colonies in Ubatuba go through

a mild seasonal bleaching during the warmer months in the summer and recover during

the remainder of the year when temperatures are lower (Fig. 10 and 11). The

phenomenon of coral bleaching is defined by the loss of Symbiodinium cells, resulting in

accessory pigment breakdown and subsequent tissue loss, leaving only the white

skeleton (Iglesias-Prieto et al., 1992; Hoegh-Guldberg, 1999; Fitt et al., 2001). The main

cause of bleaching is elevated seawater temperature (Fitt et al., 2001) and data collected

at Ubatuba revealed a yearly oscillation of 15°C, which is significantly high for

hermatypic corals. Another experiment did monitor seasonal variation in Symbiodinium

33

concentrations in M. hispida and other two species of endemic corals at Picãozinho

(Paraíba State, Northeast Brazil), but no bleaching was detected (Costa et al., 2005).

Several parameters such as temperature, salinity, nutrients, light availability and

hydrodynamics define the geographical limits for the occurrence of scleractinian corals

(Smith and Buddmeiro 1992; Kleypas 1997; Kleypas et al., 1999). The distribution limit

of Mussismilia hispida is the state of São Paulo (approx. 24° S) (Francini et al., 2002), and

at Ubatuba (S 23º 26' 02"; W 45º 04' 16") they are considered to be marginal to their

distribution (Leão et al., 2003). M. hispida is the only reef-building representative of the

order Scleractinia in the region, and it has been reported as capable of spawning in this

area (Francini et al., 2002). The heterotrophic feeding by M. hispida may be an important

adaptation to less than optimal conditions, as they may resort to this nutritional mode

when photosynthesis is suppressed. It has been reported that, after prolonged bleaching

events, corals without significant input of heterotrophic carbon such as Porites are

expected to be more susceptible to mortality than corals such as Montipora capitata,

which presents high rates of heterotrophic feeding (Grottoli et al., 2006). This fact

suggests that Mussismilia hispida has an ecological advantage over other species due to

its ability to feed on zooplankton and survive during prolonged periods of bleaching

(Grottoli et al., 2006). Furthermore, it is feasible that due to its heterotrophic capacity,

M. hispida has evolved and adapted to survive and reproduce under suboptimal

conditions, expanding its distribution. These adaptations may prove crucial in view of

current climate change projections, since this species may expand its distribution to

areas previously occupied by less tolerant species. In the future, the disproportional

presence of M. hispida could maybe used as a proxy for coral habitat degradation.

Our major findings show that M. hispida is able to frequently shift between

predominantly autotrophic and predominantly heterotrophic, especially when adverse

climate events occur. Furthermore, we have validated the use of specific fatty acid

trophic markers as proxies for these feeding modes in corals. This opens perspectives

for further studies of benthic trophic ecology in coral reefs. The contribution of this work

also includes the first yearlong monitoring of the feeding behavior in a hermatypic coral

in the South Atlantic, with the occurrence and monitoring of a mild bleaching event.

34

LITERATURE CITED

ALVES DE LIMA, L., MIGLIOLO, L., BARREIRO E CASTRO, C., DE OLIVEIRA PIRES, D., LÓPEZ

ABARRATEGUI, C., FERREIRA GONCALVES, E., MARIA VASCONCELOS, I., JOSE, C. T. A.

O., OTERO-GONZALEZ, A. J., OCTAVIO, L. F., AND CAMPOS DIAS, S. (2013).

Identification of a novel antimicrobial peptide from Brazilian coast coral Phyllogorgia

ilatata. Protein and peptide letters, 20(10), 1153-1158.

ANTHONY, K. R. N., AND FABRICIUS, K. E. (2000). Shifting roles of heterotrophy and

autotrophy in coral energetics under varying turbidity. Journal of experimental

marine biology and ecology, 252(2), 221-253.

ANTHONY, K. R. N., AND HOEGH‐GULDBERG, O. (2003a). Variation in coral

photosynthesis, respiration and growth characteristics in contrasting light

microhabitats: an analogue to plants in forest gaps and understoreys? Functional

Ecology, 17(2), 246-259.

ANTHONY, K. R. N., AND HOEGH-GULDBERG, O. (2003b). Kinetics of photoacclimation in

corals. Oecologia, 134(1), 23-31.

BACHOK, Z., MFILINGE, P., AND TSUCHIYA, M. (2006). Characterization of fatty acid

composition in healthy and bleached corals from Okinawa, Japan. Coral Reefs, 25(4),

545-554.

BELLWOOD, D. R., AND WAINWRIGHT, P. C. (2002). The history and biogeography of

fishes on coral reefs. Coral reef fishes: dynamics and diversity in a complex

ecosystem, 5-32.

BELLWOOD, D. R., HUGHES, T. P., FOLKE, C., AND NYSTRÖM, M. (2004). Confronting the

coral reef crisis. Nature, 429(6994), 827-833.

BISHOP, D. G., AND KENRICK, J. R. (1980). Fatty acid composition of symbiotic

zooxanthellae in relation to their hosts. Lipids, 15(10), 799-804.

BLIGH, E. G., AND DYER, W. J. (1959). A rapid method of total lipid extraction and

purification. Canadian journal of biochemistry and physiology, 37(8), 911-917.

BURKE, L., REYTAR, K., SPALDING, M., AND PERRY, A. (2011). Reefs at risk revisited.

World Resouces Institue: Washington DC. ISBN 98-1-56973-762.0 115 pp. Available

at: https://goo.gl/Vu8t3N. Accessed in: 16/09/2016.

CALADO, L. (2006). Dinâmica da interação da atividade de meso-escala da Corrente do

Brasil com o fenômeno da ressurgência costeira ao largo de Cabo Frio e Cabo de Sao

Tome, RJ (Doctoral dissertation, Universidade de São Paulo).

35

CANTIN, N. E., COHEN, A. L., KARNAUSKAS, K. B., TARRANT, A. M., AND MCCORKLE, D.

C. (2010). Ocean warming slows coral growth in the central Red Sea.

Science, 329(5989), 322-325.

CASTRO FILHO, B. M., MIRANDA, L.B. AND MYAO, S.Y. (1987). Considerações

hidrográficas na plataforma continental ao largo de Ubatuba: variações sazonais e

em média escala. Boletim do Instituto Oceanográfico, 35: 135-151.

CESAR, H., BURKE, L., AND PET-SOEDE, L. (2003). The economics of worldwide coral reef

degradation. Cesar Environmental Economics Consulting. Technical Report.

CINEL, B., ROBERGE, M., BEHRISCH, H., VAN OFWEGEN, L., CASTRO, C. B., AND

ANDERSEN, R. J. (2000). Antimitotic Diterpenes from Erythropodium c aribaeorum

Test pharmacophore Models for Microtubule Stabilization. Organic letters, 2(3), 257-

260.

CLARK, K. B., AND JENSEN, K. R. (1982). Effects of temperature on carbon fixation and

carbon budget partitioning in the zooxanthellal symbiosis of Aiptasia pallida

(Verrill). Journal of Experimental Marine Biology and Ecology, 64(3), 215-230.

CONNELL, J. H. (1978). Diversity in tropical rain forests and coral reefs.

Science, 199(4335), 1302-1310.

CONNELL, J.H., 1997. Disturbance and recovery of coral assemblages. Coral Reefs 16,

s101–s113.

CONNELL, J.H., HUGHES, T.P., AND WALLACE, C.C. (1997). A 30 year study of coral

abundance, recruitment, and disturbance at several scales in space and time.

Ecological Monographs, 67, 461–488.

CONNOLLY, S. R., LOPEZ-YGLESIAS, M. A., AND ANTHONY, K. R. N. (2012). Food

availability promotes rapid recovery from thermal stress in a scleractinian coral. Coral

Reefs, 31(4), 951-960.

COSTA, C. F., SASSI, R., AND AMARAL, F. D. (2005). Annual cycle of symbiotic

dinoflagellates from three species of scleractinian corals from coastal reefs of

northeastern Brazil. Coral Reefs, 24(2), 191-193.

COSTANZA, R., D'ARGE, R., DE GROOT, R., FABER, S., GRASSO, M., HANNON, B., AND

RASKIN, R. G. (1997). The value of the world's ecosystem services and natural capital.

The Globalization and Environment Reader, 117.

CRABBE, M.J.C., MENDES, J.M., AND WARNER, G.F. (2002). Lack of recruitment of

nonbranching corals in Discovery Bay is linked to severe storms. Bulletin of Marine

Science, 70, 939–945.

36

CRABBE, M.J.C., WALKER, E.L.L., AND STEPHENSON, D.B. (2008). The impact of weather

and climate extremes on coral growth. In: Diaz, H., Murnane, R. (Eds.), Climate

Extremes and Society. Cambridge University Press, Cambridge, UK, 165– 188.

CROSSLAND, C. J. (1987). In situ release of mucus and DOC-lipid from the corals Acropora

variabilis and Stylophora pistillata in different light regimes. Coral reefs, 6(1), 35-42.

CROSSLAND, C. J., BARNES, D. J., AND BOROWITZKA, M. A. (1980). Diurnal lipid and

mucus production in the staghorn coral Acropora acuminata. Marine Biology,60(2-3),

81-90.

DALSGAARD, J., JOHN, M. S., KATTNER, G., MÜLLER-NAVARRA, D., AND HAGEN, W.

(2003). Fatty acid trophic markers in the pelagic marine environment. Advances in

marine biology, 46, 225-340.

DEWICK, P. M. (1997). The biosynthesis of C 5–C 25 terpenoid compounds. Natural

Product Reports, 14(2), 111-144.

DODDS, L. A., BLACK, K. D., ORR, H., AND ROBERTS, J. M. (2009). Lipid biomarkers reveal

geographical differences in food supply to the cold-water coral Lophelia pertusa

(Scleractinia). Marine Ecology Progress Series, 397, 113-124.

DOUGLAS, A. E. (2003). Coral bleaching––how and why? Marine Pollution Bulletin, 46(4),

385-392.

DUBINSKY, Z., AND FALKOWSKI, P. (2011). Light as a source of information and energy

in zooxanthellate corals. Springer Netherlands, (pp. 107-118).

EZZAT, L., TOWLE, E., IRISSON, J. O., LANGDON, C., AND FERRIER‐PAGÈS, C. (2016). The

relationship between heterotrophic feeding and inorganic nutrient availability in the

scleractinian coral T. reniformis under a short‐term temperature increase. Limnology

and Oceanography, 61(1), 89-102.

FABRICIUS, K. (1999). Tissue loss and mortality in soft corals following mass-

bleaching. Coral Reefs,18(1), 54-54.

FABRICIUS, K. E., AND DOMMISSE, M. (2000). Depletion of suspended particulate matter

over coastal reef communities dominated by zooxanthellate soft corals. Marine

Ecology Progress Series, 196, 157-167.

FALKOWSKI, P. G., DUBINSKY, Z., MUSCATINE, L., AND PORTER, J. W. (1984). Light and

the bioenergetics of a symbiotic coral. Bioscience, 34(11), 705-709.

FALTER, J. L., ATKINSON, M. J., SCHAR, D. W., LOWE, R. J., AND MONISMITH, S. G. (2011).

Short-term coherency between gross primary production and community respiration

in an algal-dominated reef flat. Coral Reefs, 30(1), 53-58.

37

FERRIER-PAGES, C., WITTING, J., TAMBUTTÉ, E., AND SEBENS, K. P. (2003). Effect of

natural zooplankton feeding on the tissue and skeletal growth of the scleractinian

coral Stylophora pistillata. Coral Reefs, 22(3), 229-240.

FIGUEIREDO, J., BAIRD, A. H., COHEN, M. F., FLOT, J. F., KAMIKI, T., MEZIANE, T., AND

YAMASAKI, H. (2012). Ontogenetic change in the lipid and fatty acid composition of

scleractinian coral larvae. Coral Reefs, 31(2), 613-619.

FITT, W. K., BROWN, B. E., WARNER, M. E., AND DUNNE, R. P. (2001). Coral bleaching:

interpretation of thermal tolerance limits and thermal thresholds in tropical

corals. Coral Reefs, 20(1), 51-65.

FRANCINI, C. L. B., CASTRO, C. B., AND PIRES, D. O. (2002). First record of a reef coral

spawning event in the western South Atlantic. Invertebrate reproduction and

development, 42(1), 17-19.

GILMOUR, J. P., SMITH, L. D., HEYWARD, A. J., BAIRD, A. H., AND PRATCHETT, M. S.

(2013). Recovery of an isolated coral reef system following severe

disturbance. Science, 340(6128), 69-71.

GLYNN, P. W., AND ENOCHS, I. C. (2011). Invertebrates and their roles in coral reef

ecosystems. In Coral reefs: an ecosystem in transition. Springer Netherlands, 273-325

GROTTOLI, A. G. (2002). Effect of light and brine shrimp on skeletal δ 13 C in the

Hawaiian coral Porites compressa: a tank experiment. Geochimica et Cosmochimica

Acta, 66(11), 1955-1967.

GROTTOLI, A. G., AND WELLINGTON, G. M. (1999). Effect of light and zooplankton on

skeletal δ13C values in the eastern Pacific corals Pavona clavus and Pavona

gigantea. Coral Reefs, 18(1), 29-41.

GROTTOLI, A. G., RODRIGUES, L. J., AND PALARDY, J. E. (2006). Heterotrophic plasticity

and resilience in bleached corals. Nature, 440(7088), 1186-1189.

HALFAR, J., GODINEZ-ORTA, L., RIEGL, B., VALDEZ-HOLGUIN, J. E., AND BORGES, J. M.

(2005). Living on the edge: high-latitude Porites carbonate production under

temperate eutrophic conditions. Coral Reefs, 24(4), 582-592.

HALLOCK, P. (1997). Reefs and reef limestones in Earth history. In: Birkeland, C (Ed.), Life

and Death of Coral Reefs. Chapman and Hall, New York, 13-42.

HODGSON, G. (1999). A global assessment of human effects on coral reefs. Marine

Pollution Bulletin, 38, 2–8.

HOEGH-GULDBERG, O. (1999). Climate change, coral bleaching and the future of the

world's coral reefs. Marine and freshwater research, 50(8), 839-866.

38

HOEGH-GULDBERG, O., MUMBY, P. J., HOOTEN, A. J., STENECK, R. S., GREENFIELD, P.,

GOMEZ, E., AND KNOWLTON, N. (2007). Coral reefs under rapid climate change and

ocean acidification. Science, 318(5857), 1737-1742.

HOOGENBOOM, M., E. BERAUD, AND C. FERRIER-PAGÈS. (2010). Relationship between

symbiont density and photosynthetic carbon acquisition in the temperate coral

Cladocora caespitosa. Coral Reefs 29: 21–29. DOI: 10.1007/s00338-009-0558-9

HOOGENBOOM, M., RODOLFO-METALPA, R., AND FERRIER-PAGÈS, C. (2010). Co-

variation between autotrophy and heterotrophy in the Mediterranean coral

Cladocora caespitosa. Journal of Experimental Biology, 213(14), 2399-2409.

HOULBREQUE, F., AND C. FERRIER-PAGES. (2009). Heterotrophy in tropical scleractinian

corals. Biological Reviews. 84: 1–17. DOI: 10.1111/j.1469-185X.2008.00058.x

HOULBRÈQUE, F., TAMBUTTÉ, E., ALLEMAND, D., AND FERRIER-PAGÈS, C. (2004).

Interactions between zooplankton feeding, photosynthesis and skeletal growth in the

scleractinian coral Stylophora pistillata. Journal of Experimental Biology, 207(9),

1461-1469.

HOULBRÈQUE, F., TAMBUTTÉ, E., AND FERRIER-PAGÈS, C. (2003). Effect of zooplankton

availability on the rates of photosynthesis, and tissue and skeletal growth in the

scleractinian coral Stylophora pistillata. Journal of Experimental Biology, 296, 145–

166.

Hughes, T. P., Baird, A. H., Bellwood, D. R., Card, M., Connolly, S. R., Folke, C., Grosberg

R., Hoegh-Guldberg, O., Jackson, J. B. C., Kleypas, J., Lough, J. M., Marshall, P.,

Nyström, M., Palumbi, S. R., Pandolfi, M., Rosen, M., AND Lough, J. M. (2003). Climate

change, human impacts, and the resilience of coral reefs. Science, 301(5635), 929-

933.

IGLESIAS-PRIETO, R., MATTA, J. L., ROBINS, W. A., AND TRENCH, R. K. (1992).

Photosynthetic response to elevated temperature in the symbiotic dinoflagellate

Symbiodinium microadriaticum in culture. Proceedings of the national Academy of

Sciences, 89(21), 10302-10305.

IVERSON, S. J., FIELD, C., DON BOWEN, W., AND BLANCHARD, W. (2004). Quantitative

fatty acid signature analysis: a new method of estimating predator diets. Ecological

Monographs, 74(2), 211-235.

JOHANNES, R. E., COLES, S. L., AND KUENZEL, N. T. (1970). The role of zooplankton in the

nutrition of some scleractinian corals. Limnology and oceanography, 15(4), 579-586.

KLEYPAS, J. A. (1997). Modeled estimates of global reef habitat and carbonate

production since the last glacial maximum. Paleoceanography, 12(4), 533-545.

39

KLEYPAS, J. A., MCMANUS, J. W., AND MEÑEZ, L. A. (1999). Environmental limits to coral

reef development: where do we draw the line? American Zoologist, 39(1), 146-159.

KRUEGER, T., HAWKINS, T. D., BECKER, S., PONTASCH, S., DOVE, S., HOEGH-GULDBERG,

O., AND DAVY, S. K. (2015). Differential coral bleaching—Contrasting the activity and

response of enzymatic antioxidants in symbiotic partners under thermal

stress. Comparative Biochemistry and Physiology Part A: Molecular AND Integrative

Physiology, 190, 15-25.

LALLI, C. M. AND PARSONS, T. R. 2004. Biological oceanography. An introduction. 2nd

edition. Elsevier Butterworth–Heinemann, 314.

Leãoa, Z. M., Kikuchi, R. K., AND Testa, V. (2003). Corals and coral reefs of Brazil. In: Latin

American Coral Reefs. Elsevier Science B.V. 9-52.

LEVAS, S. J., GROTTOLI, A. G., HUGHES, A., OSBURN, C. L., AND MATSUI, Y. (2013).

Physiological and biogeochemical traits of bleaching and recovery in the mounding

species of coral Porites lobata: implications for resilience in mounding corals. PloS

one, 8(5), e63267.

LOPES, R. M. (2013). Variação temporal e crescimento do zooplâncton no litoral norte

de São Paulo, com ênfase em estágios imaturos de copépodes(Doctoral dissertation,

Universidade de São Paulo).

MAHIQUES, M. D. (1995). Dinâmica sedimentar atual nas enseadas da região de

Ubatuba, Estado de São Paulo. Boletim do Instituto Oceanográfico, 43(2), 111-122.

MALLELA, J. AND CRABBE, M. J. C. (2009). Hurricanes and coral bleaching linked to

changes in coral recruitment in Tobago. Marine Environmental Research, 68(4), 158-

162.

CRABBE, M.J.C., MENDES, J.M., WARNER, G.F., (2002). Lack of recruitment of

nonbranching corals in Discovery Bay is linked to severe storms. Bulletin of Marine

Science, 70, 939–945.

MALLELA, J. (2007). Coral reef encruster communities and carbonate production in

cryptic and exposed coral reef habitats along a gradient of terrestrial disturbance.

Coral Reefs 26, 775–785.

MCCLANAHAN, T. R., MUTHIGA, N. A., KAMUKURU, A. T., MACHANO, H., AND KIAMBO,

R. W. (1999). The effects of marine parks and fishing on coral reefs of northern

Tanzania. Biological Conservation, 89(2), 161-182.

MILLER, M. W. (1995). Growth of a temperate coral: effects of temperature, light, depth,

and heterotrophy. Marine Ecology Progress Series, 122, 217-225.

40

MOBERG, F., AND FOLKE, C. (1999). Ecological goods and services of coral reef

ecosystems. Ecological economics, 29(2), 215-233.

MUSCATINE, L., AND PORTER, J. W. (1977). Reef corals: mutualistic symbioses adapted

to nutrient-poor environments. Bioscience, 27(7), 454-460.

MUSCATINE, L., AND WEIS, V. (2013). Productivity of zooxanthellae and

biogeochemical. Primary Productivity and Biogeochemical Cycles in the Sea, 43, 257.

NAPOLITANO, G. E., POLLERO, R. J., GAYOSO, A. M., MACDONALD, B. A., AND

THOMPSON, R. J. (1997). Fatty acids as trophic markers of phytoplankton blooms in

the Bahia Blanca estuary (Buenos Aires, Argentina) and in Trinity Bay (Newfoundland,

Canada). Biochemical Systematics and Ecology, 25(8), 739-755.

SCHNELL, R. C. (2004). Climate Monitoring and Diagnostics Laboratory summary. Report,

2002–2003. In: National Oceanic Atmospheric Administration, 27, 174.

OLIVOTTO I., ROLLO A., SULPIZIO R., AVELLA M., TOSTI L., CARNEVALI O. (2006) Breeding

and rearing the Sunrise Dotty back Pseudo chromis flavivertex: the importance of live

prey enrichment during larval development. Aquaculture, 255, 480–487.

OSINGA, R., SCHUTTER, M., GRIFFIOEN, B., WIJFFELS, R. H., VERRETH, J. A., SHAFIR, S.,

AND LAVORANO, S. (2011). The biology and economics of coral growth. Marine

Biotechnology, 13(4), 658-671.

PALARDY, J. E., GROTTOLI, A. G., AND MATTHEWS, K. A. (2005). Effects of upwelling,

depth, morphology and polyp size on feeding in three species of Panamanian

corals. Marine Ecology Progress Series, 300, 79-89.

PALARDY, J. E., GROTTOLI, A. G., AND MATTHEWS, K. A. (2006). Effect of naturally

changing zooplankton concentrations on feeding rates of two coral species in the

Eastern Pacific. Journal of Experimental Marine Biology and Ecology, 331(1), 99-107.

PAPINA, M., MEZIANE, T., AND VAN WOESIK, R. (2003). Symbiotic zooxanthellae provide

the host-coral Montipora digitata with polyunsaturated fatty acids. Comparative

Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 135(3), 533-

537.

PAPINA, M., MEZIANE, T., AND VAN WOESIK, R. (2007). Acclimation effect on fatty acids

of the coral Montipora digitata and its symbiotic algae. Comparative Biochemistry

and Physiology Part B: Biochemistry and Molecular Biology, 147(4), 583-589.

PERSSON, J., AND VREDE, T. (2006). Polyunsaturated fatty acids in zooplankton:

variation due to taxonomy and trophic position. Freshwater Biology, 51(5), 887-900.

ROBERTS, C.M., MCCLEAN, C.J., VERON, J.E.N., HAWKINS, J.P., ALLEN, G.R., MCALLISTER,

D.E., MITTERMEIER, C.G., SCHUELER, F.W., SPALDING, M., WELLS, F., VYNNE, C., AND

41

WERNER, T.B. (2002). Marine biodiversity hotspots and conservation priorities for

tropical reefs. Science, 295, 1280–1284.

RODRIGUES, L. J., AND GROTTOLI, A. G. (2006). Calcification rate and the stable carbon,

oxygen, and nitrogen isotopes in the skeleton, host tissue, and zooxanthellae of

bleached and recovering Hawaiian corals. Geochimica et Cosmochimica Acta, 70(11),

2781-2789.

RODRIGUES, L. J., AND GROTTOLI, A. G. (2007). Energy reserves and metabolism as

indicators of coral recovery from bleaching. Limnology and oceanography, 52(5),

1874-1882.

RUPPERT, E. E., FOX, R. S. BARNES, R. D. (2004). Invertebrate Zoology, 7th edition.

Cengage Learning. 132–137. ISBN 978-81-315-0104-7.

SALVAT, B. (1992). Coral reefs: a challenging ecosystem for human societies. Global

Environmental Change, 2(1), 12-18.

SARGENT, J.R., BELL MV, HENDERSEN, R.J., AND TOCHER, D.R. (1990) Polyunsaturated

fatty acids in marine and terrestrial food webs. In: Mellinger, J. (eds) Animal nutrition

and transport processes, 1, Nutrition in wild and domestic animals. Comparative

physiology. Karger, Basel, 11–23.

SEBENS, K. P., HELMUTH, B., CARRINGTON, E., AND AGIUS, B. (2003). Effects of water

flow on growth and energetics of the scleractinian coral Agaricia tenuifolia in

Belize. Coral Reefs, 22(1), 35-47.

SEBENS, K. P., VANDERSALL, K. S., SAVINA, L. A., AND GRAHAM, K. R. (1996). Zooplankton

capture by two scleractinian corals, Madracis mirabilis andMontastrea cavernosa, in

a field enclosure. Marine Biology, 127(2), 303-317.

SHEPPARD, C. R., DAVY, S. K., AND PILLING, G. M. (2009). The biology of coral reefs. OUP

Oxford.

SMITH, S. V., AND BUDDEMEIER, R. W. (1992). Global change and coral reef

ecosystems. Annual Review of Ecology and Systematics, 23, 89-118.

SOROKIN, Y. I. (2013). Coral reef ecology. Springer Science AND Business Media, (102).

STAT, M., CARTER, D., AND HOEGH-GULDBERG, O. (2006). The evolutionary history of

Symbiodinium and scleractinian hosts: symbiosis, diversity, and the effect of climate

change. Perspectives in Plant Ecology, Evolution and Systematics, 8(1), 23-43.

TITLYANOV, E. A., TITLYANOVA, T. V., YAMAZATO, K., AND VAN WOESIK, R. (2001).

Photo-acclimation dynamics of the coral Stylophora pistillata to low and extremely

low light. Journal of Experimental Marine Biology and Ecology, 263(2), 211-225.

42

TREIGNIER, C., GROVER, R., FERRIER-PAGES, C., AND TOLOSA, I. (2008). Effect of light

and feeding on the fatty acid and sterol composition of zooxanthellae and host tissue

isolated from the scleractinian coral Turbinaria reniformis. Limnology and

Oceanography, 53(6), 2702-2710.

UNEP - UNITED NATIONS ENVIRONMENT PROGRAM (2003): Conventions and Coral

Reef, Fourteen Multilateral Environment Agreements, Programmes, Partnerships

and Networks Relevant to the Protection and Conservation of Coral Reef, and the

World Summit on Sustainable Development Plan of Implementation. Produced by the

UNEP Coral Reef Unit in collaboration with the WWF Coral Reef Advocacy Initiative.

Available at - http://www.unep.org/annualreport/2013/landing.asp (Last accessed

in: 06/21/2016).

VENN, A. A., TAMBUTTÉ, E., HOLCOMB, M., LAURENT, J., ALLEMAND, D., AND

TAMBUTTÉ, S. (2013). Impact of seawater acidification on pH at the tissue–skeleton

interface and calcification in reef corals. Proceedings of the National Academy of

Sciences, 110(5), 1634-1639.

VOLKMAN, J. K., BARRETT, S. M., AND BLACKBURN, S. I. (1999). Eustigmatophyte

microalgae are potential sources of C 29 sterols, C 22–C 28 n-alcohols and C 28–C 32

n-alkyl diols in freshwater environments. Organic Geochemistry, 30(5), 307-318.

WABNITZ C., TAYLOR M., GREEN E., RAZAK T. (2003) From Ocean to Aquarium.

Cambridge: UNEP–WCMC.

WILD, C., HUETTEL, M., KLUETER, A., KREMB, S. G., RASHEED, M. Y., AND JØRGENSEN, B.

B. (2004). Coral mucus functions as an energy carrier and particle trap in the reef

ecosystem. Nature, 428(6978), 66-70.

WILKINSON, C. (2008). Status of coral reefs of the world: 2008. Global Coral Reef

Monitoring Network and Reef and Rainforest Research Centre, Townsville, Australia.

WILSON, S. K., BURNS, K., AND CODI, S. (2001). Sources of dietary lipids in the coral reef

blenny Salarias patzneri. Marine Ecology Progress Series, 222, 291-296.

WPACFIN (Western Pacific Fishery Information Network). NOAA-National Marine

Fisheries Service, Pacific Islands Fisheries Science Center, 2570 Dole Street, Honolulu,

96822–2396. Available at: http://www.pifsc.noaa.gov/wpacfin (Last accessed

07/30/2016).

ZHUKOVA, N. V., AND TITLYANOV, E. A. (2003). Fatty acid variations in symbiotic

dinoflagellates from Okinawan corals. Phytochemistry, 62(2), 191-195.