Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

Settlement and post-settlement survival of Orbicella annularis and

Orbicella faveolata (Scleractinia: Merulinidae) on substrates with coatings

Laura C. Arango-Carvajal1*; https://orcid.org/0000-0002-9554-7425

Lizette I. Quan-Young2; https://orcid.org/0000-0002-2393-7328

Adrián Villegas-Jiménez3; https://orcid.org/0009-0001-2167-0367

Anastazia T. Banaszak4; https://orcid.org/0000-0002-6667-3983

1. Programa de Biología, Facultad de Ciencias y Biotecnología, Universidad CES, Calle 10A No. 22 – 04, Medellín,

Colombia; arangoc.laura@uces.edu.co (*Correspondence)

2. Grupo de Investigación Biología CES, Facultad de Ciencias y Biotecnología, Universidad CES, Calle 10A No. 22 – 04,

Medellín, Colombia; lquan@ces.edu.co

3. WhiteRock Research & Consulting, 15 Avenida Sur No. 1148, Cozumel, Q. Roo 77664, México;

avillegas@wrcapitalnatural.com

4. Unidad Académica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnología, Universidad Nacional

Autónoma de México, Puerto Morelos, Q. Roo 77580, México; banaszak@cmarl.unam.mx

Received 30-IX-2022. Corrected 07-III-2023. Accepted 13-III-2023.

ABSTRACT

Introduction: One of the main bottlenecks in restoration projects based on sexual reproduction is post-settle-

ment survival, mainly due to competition for substrate with fleshy algae and predation. Therefore, substrates of

different shapes and materials have been created and tested, seeking to optimize these processes with attractive

surfaces for the larvae and structures where the recruits are protected from predation, and competition is reduced.

Objective: To improve settlement and post-settlement survival of two important Caribbean reef-building corals,

using different coatings on substrates.

Methods: To determine whether substrate coatings properties are favourable to larval settlement in Orbicella

annularis, and O. faveolata, collected in Puerto Morelos, Mexican Caribbean, we evaluated their settlement for

three weeks on six coatings with a combination of properties. Each coating was designed to provide a combina-

tion of two out of three properties: 1) water repellence (hydrophobicity), 2) phosphorescence-based colour, and

3) mineral-enriched surface chemistry. In a separate experiment larvae settlement was tested using coatings with

a single property. Finally, we determined the post-settlement survival of O. annularis and O. faveolata on the

different coatings for seven weeks.

Results: The combination of high hydrophobicity and light blue phosphorescent microparticles and high hydro-

phobicity and red-orange phosphorescent microparticles resulted in a higher settlement of O. annularis and O.

faveolata when compared with other coatings (30.8 - 66.7 % higher). No significant differences were found in

the number of larval settled when the water-repellence and the phosphorescence-based were evaluated indepen-

dently. Post-settlement survival time on substrates was low, with a maximum of 34 days after settlement for O.

annularis and 42 days for O. faveolata.

Conclusions: In terms of the larval settlement, the combination of the coatings properties appears to play an

essential role in the choice of microhabitat for both O. annularis and O. faveolata. But individually these prop-

erties did not generate an advantage in the larval settlement. Moreover, some chemical components associated

with the coatings may be counterproductive to the survival of the polyps over time.

Key words: reef restoration; sexual reproduction; scleractinian corals; reef conservation; coral larvae.

https://doi.org/10.15517/rev.biol.trop..v71iS1.54864

SUPPLEMENT

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

INTRODUCTION

Coral reefs are characterized by high bio-

diversity, hosting 32 % of all known marine

species and provide essential ecosystem ser-

vices that contribute to the well-being of mil-

lions of people (Fisher et al., 2015; Knowlton

et al., 2010; Woodhead et al., 2019). They are

considered endangered ecosystems (Carpen-

ter et al., 2008) and, most importantly, one

of the most sensitive ecosystems to climate

change (Riegl et al., 2009). Since 1970, coral

cover in the Caribbean has decreased by 80

%, mainly due to the high mortality of reef-

building corals, which has led to a phase-shift

from coral to algae, and a loss of ecological

functionality, as the result of both natural and

anthropogenic impacts (Álvarez-Filip et al.,

2022; Arias-González et al., 2017; Aronson &

Precht, 2001; Jackson et al., 2014; Rioja-Nieto

& Álvarez-Filip, 2019). This has brought about

the loss of recruitment for the main reef-build-

ing corals (Edmunds & Elahi, 2007; Edmunds

et al., 2011; Hernández-Delgado et al., 2014;

Quinn & Kojis, 2005; Williams et al., 2008),

which include the species Orbicella annularis

(Ellis & Solander, 1786) and Orbicella faveo-

lata (Ellis & Solander, 1786).

Larval settlement as well as survival and

growth of coral species, are integral to the

ongoing maintenance of coral populations and

necessary for the recovery of coral reefs (Rit-

son-Williams et al., 2009). Many factors can

impact these processes, such as the abundance

of fleshy algae, competition with other ben-

thic invertebrates, predation, and sedimentation

RESUMEN

Asentamiento y supervivencia post-asentamiento de Orbicella annularis y Orbicella faveolata

(Scleractinia: Merulinidae) sobre sustratos con recubrimientos.

Introducción: Uno de los principales cuellos de botella en proyectos de restauración basada en reproducción

sexual es la supervivencia de las larvas posterior al asentamiento, principalmente por la competencia por el sus-

trato con algas filamentosas y la depredación. Por ello, se han creado y analizado sustratos de diferentes formas

y materiales, buscando optimizar estos procesos con superficies atrayentes para las larvas, y estructuras donde

los reclutas se encuentran protegidos de la depredación y se disminuya la competencia.

Objetivo: Mejorar el asentamiento y la supervivencia de dos importantes corales formadores de arrecifes del

Caribe, utilizando diferentes recubrimientos en sustratos.

Métodos: Para determinar si las propiedades de la superficie del sustrato son favorables para el asentamiento de

larvas de Orbicella annularis y O. faveolata, recolectadas en Puerto Morelos, Caribe mexicano, evaluamos su

asentamiento durante tres semanas en seis recubrimientos con una combinación de propiedades. Cada recubri-

miento fue diseñado para proporcionar una combinación de dos de tres propiedades: 1) repelencia al agua (hidro-

fobicidad), 2) fosforescencia y 3) química superficial enriquecida con minerales. En un experimento separado se

evaluó el asentamiento de larvas en sustratos con recubrimientos de una sola propiedad. Finalmente, se determinó

la supervivencia posterior al asentamiento de O. annularis y O. faveolata sobre los diferentes recubrimientos

durante siete semanas.

Resultados: La combinación de alta hidrofobicidad y micropartículas fosforescentes azules y alta hidrofobicidad

y micropartículas fosforescentes rojo-naranja dio como resultado un mayor asentamiento de O. annularis y O.

faveolata en comparación con otros recubrimientos (30.8 - 66.7 % mayor). No se encontraron diferencias signi-

ficativas en el número de larvas asentadas cuando se evaluaron de forma independiente la repelencia al agua y

la fosforescencia. El tiempo de supervivencia posterior al asentamiento en los sustratos fue bajo, con un máximo

de 34 días después del asentamiento para O. annularis y 42 días para O. faveolata.

Conclusiones: En el asentamiento de larvas, la combinación de las propiedades del recubrimiento parece desem-

peñar un papel importante en la elección del microhábitat tanto para O. annularis como para O. faveolata. Pero

de forma individual estas propiedades no generaron una ventaja en el asentamiento larvario. Además, algunos

componentes químicos asociados con los recubrimientos pueden ser contraproducentes para la supervivencia de

los pólipos a lo largo del tiempo.

Palabras clave: restauración de arrecifes; reproducción sexual; corales escleractinios; conservación de arrecifes;

larvas de coral.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

(Pizarro et al., 2007). To assist in the recruit-

ment of corals, restoration strategies have

become more critical over the past decades

and have involved culturing coral settlers from

gametes to improve the genetic diversity of

coral population (Baums et al., 2019). Howev-

er, one of the significant bottlenecks in this pro-

cess is post-settlement survival and coral settler

growth (Ritson-Williams et al., 2009), mainly

due to competition with fleshy algae and pre-

dation (Edwards & Gomez, 2007), but also

include important abiotic factors such as water

clarity, sedimentation, nutrient enrichment,

salinity, and temperature (Richmond, 1997).

In controlled conditions (Ex-situ) settle-

ment may be enhanced by using substrates with

surfaces containing specific larvae-attracting

attributes, including varying colour phospho-

rescence properties. Phosphorescence is a spe-

cific type of photoluminescence whereby, once

the light has been absorbed, it is emitted and

continues to be emitted even after excitation

has ceased (Valeur & Berberan-Santos, 2011).

It is a property that can generate micro-envi-

ronments of coloured light in otherwise dark

conditions, sometimes preferred by certain spe-

cies as a condition for settlement (Mason et al.,

2011; Strader et al., 2015).

Post-settlement survival may also be

enhanced by using substrates with surface

properties that may promote a reduction in

competition, nutrient supply, and coral skeleton

growth. This can be achieved by engineer-

ing substrate coatings with different chemical

properties and hydrophobicity (Levenstein et

al., 2022). Hydrophobicity can play an impor-

tant role in reducing competition with algae

from the genus Ulva, as it can affect algal

spore adhesion and the strength of the adhesion

(Chaudhury et al., 2006; Finlay, 2002; Fletcher

& Callow, 1992). On the other hand, the

presence of hydroxyapatite on the substrate’s

surface can be a source of phosphate released

as the hydroxyapatite potentially dissolves in

low phosphate ion concentration scenarios in

seawater (Atlas & Pytkowicz, 1977; Stumm &

Morgan, 1996). This nutrient may be taken up

by symbiotic dinoflagellates and converted into

essential organic molecules (Ezzat et al., 2016).

Similarly, the presence of calcium carbonate on

a substrate’s surface provides a source of car-

bonate and calcium ions, the building blocks of

aragonite-based coral skeletons, which can be

rapidly released upon CaCO3(s) dissolution in

response to local variations in seawater chem-

istry, affecting CaCO3(s) saturation state, such

as acidity build up in the vicinity of the coral

tissue-seawater interface (Venn et al., 2013).

The research presented here aims to

investigate the synergistic effects of specific

physical-chemical properties (hydrophobicity,

phosphorescence and mineral-enriched sur-

face chemistry) on settlement as well as post-

settlement survival for O. annularis, and O.

faveolata from Puerto Morelos, Mexico.

MATERIALS AND METHODS

Substrate fabrication: In 2019, coatings

were applied to commercially available floor

tiles to test a combination of properties and

their potential effect on settlement and post-

settlement survival of coral larvae. Each coat-

ing was designed to provide a combination of

two out of three properties: 1) water repellence

(as determined by its degree of hydrophobic-

ity), 2) phosphorescence-based colour, and

3) mineral-enriched surface chemistry. The

ceramic floor tiles (Interceramic©, Mexico 5

cm × 5 cm × 5 mm) were used as the sub-

strate to which six sets of coatings (C1 to C6)

were applied (Table 1). The tile surfaces were

roughened using water-based sandpaper to

improve adhesion before applying the coat-

ing. A hydrophobic polyester resin coating

served as a base for the application of micro or

nanoparticles and was applied to all tiles except

C6. Tiles C1-C3 contained microparticles of

strontium aluminate salts with varying relative

concentrations of rare earth elements europium

and trivalent dysprosium (SrxEuyDyzAl2O4),

each yielding specific colour phosphorescent

properties (Distribuidora Química Textil S.A.

de C.V., Mexico): light blue (C1), red-orange

(C2) and turquoise (C3). Phosphorescent

microparticles in tiles C2 also contained a

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

coumarin-type red-emitting fluorescent pig-

ment. On tiles C4, 60 nm hydroxyapatite

nanoparticles [Ca10(PO4)6(OH)2, 96 % purity]

(MK Impex Corp., Canada, MKN-HXAP-060)

were applied, whereas 80 nm nanoparticles

of amorphous calcium carbonate [CaCO3, >

98 % purity] (MK Impex Corp., Canada,

MKN-CaCO3-080) were applied to tiles C5

and C6. A commercially available superhydro-

phobic polysiloxane-based coating (Eronde,

USA, 9HMR-FIX) was applied on the dry

surface of the polyester resin of tiles C1, C2,

and C3. In the case of C6, the superhydropho-

bic coating was applied directly onto the tile

surface and served as a base for applying the

CaCO3 nanoparticles.

In 2021, we evaluated the individual prop-

erties of coatings on settlement preference

of Orbicella spp. larvae to determine if it is

determined by a combination of properties or

by an individual property. We focused on sub-

strates C1 and C2 because they elicited greater

settlement compared to the other tiles that were

tested. The same brand of ceramic floor tiles

from the previous experiment was used. We

prepared six treatments (T1 to T6, Table 1).

All the treatments had a hydrophobic polyester

resin coating. For treatment T1 a superhydro-

phobic polysiloxane-based coating (same as the

previous experiment) was applied. Treatments

T2 and T3 contained microparticles of stron-

tium aluminate salts with light blue and red-

orange colours, respectively. Treatment T4 had

a hydrophobic polyester resin coating applied.

White paint was added to T5 and T6 consisted

of the bare ceramic floor tile without coatings.

Substrate conditioning: Ten replicates of

each treatment (T1 to T6) were placed in flow-

through seawater aquaria (480 L) for seven

weeks to pre-condition them and allow biofilm

formation (Sneed et al., 2014). Sea water was

filtered to 1 µm and set to the following condi-

tions: temperature 27–28.5 °C, salinity 35–36

psu, pH 7.9–8.3. Concentrations of ammonia,

nitrite, and nitrate were undetectable using

standard methods (Grasshoff et al., 1999).

Therefore, the remaining ten substrates of

each treatment were maintained without condi-

tioning. Substrate conditioning was evaluated

for Orbicella spp. in 2021 (Table 2). It was

not tested in 2019, because we did not have

enough replicates.

TABLE 1

Properties and characteristics of substrate coatings.

Substrate

White

paint

Hydrophobic coatings Characteristics of micro or nanoparticles

Intermediate

hydrophobicity

polyester resin

Superhydrophobic

polysiloxane

coating

Size

particles Properties

C1 No Ye s Ye s Micro Light blue phosphorescent

C2 No Ye s Ye s Micro Red-orange phosphorescent

C3 No Ye s Ye s Micro Turquoise phosphorescent

C4 No Ye s No Nano Hydroxyapatite

C5 No Ye s No Nano Amorphous calcium carbonate

C6 No No Ye s Nano Amorphous calcium carbonate

T1 Ye s Ye s Ye s No No

T2 Ye s Ye s No Micro Light blue phosphorescent

T3 Ye s Ye s No Micro Red-orange phosphorescent

T4 Ye s Ye s No No No

T5 Ye s No No No No

T6 No No No No No

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

Collection of gametes, assisted fertiliza-

tion, and care of embryos: One week after the

full moon in September 2019, gamete bundles

were collected from four colonies of Orbicella

annularis from Jardines Reef (20°49’53.0” N,

86°52’28.2” W), as well as three colonies of O.

faveolata from La Bocana Reef (20°52’25.7”

N, 86°51’04.2” W). In September 2021 gamete

bundles were collected from 10 colonies of

O. annularis and two colonies of O. faveolata

from Jardines Reef. The gametes were col-

lected using nets that concentrated the gamete

bundles into small plastic containers at the

top of the nets. These reefs are located in the

Puerto Morelos Reef National Park, Mexican

Caribbean. Afterwards, all the gametes were

transported to the laboratory, where gamete

predators (crustaceans, polychaetes, and fish

larvae) were removed using transfer pipettes,

and gametes from the different colonies of

the same species were mixed in a single 6 L

container to assist fertilization. Subsequently,

excess sperm was removed, and the developing

embryos were transferred to a culture system,

where they developed into planula larvae.

Choice experiment: Three days after

spawning, 6 000 O. annularis larvae were

distributed evenly into five replicate sets of 6 L

plastic containers (1 200 larvae per container).

Each plastic container had two substrates of

each type of treatment (C1 to C6). The same

experimental design was repeated for O. faveo-

lata (Table 2). The settlement was recorded on

days eight, 13, and 18 after spawning. One day

after the last count of the substrates, those with

O. annularis and O. faveolata primary polyps

were transferred to flow-through aquaria with

access to natural light, food, and symbionts.

Substrates were scored for survival 13, 20, 27,

34, and 41 days after settlement.

Three days after spawning, 2 000 O. annu-

laris larvae were distributed evenly into ten

replicate sets of 2 L plastic containers (200

larvae per container). Each container had six

substrate coatings (T1 to T6). Five containers

had conditioned substrates, and five contain-

ers had unconditioned substrates (Table 2).

On days eight, 13, and 18 after spawning, O.

annularis settlement was recorded. One day

after the last count, substrates with O. annu-

laris primary polyps were transferred to flow-

through aquaria. The substrates were scored for

survival seven, 14, 21, 28, 35, and 42 days after

settlement. The same experimental setup and

methodology were used for O. faveolata.

Statistical analysis: A generalized lin-

ear model (GLM) with a negative binomial

distribution and a logit link was used to anal-

yse settlement in the O. annularis and O.

faveolata choice experiment between substrate

coatings with combinations of properties (C1

to C6, 6 levels) and in the O. annularis and

O. faveolata choice experiment to determine

the effect of substrate coatings with a single

property (T1 to T6, six levels) and substrate

conditioning (Conditioned and unconditioned,

two levels). The models are selected by the

Bayesian Information Criterion (BIC). Model

TABLE 2

Experimental design of coatings with combinations of properties (2019) and coatings with a single property (2021).

Species, year Containers Number of substrates Larvae

per ml

Variables

Coatings Conditioning

Treatments Replicates

per treatment Treatments Replicates per

treatment

O. annularis -

O. faveolata 2019

5 (6 L) 12 substrates per

container, 2 per coating

0.2 C1 to C6 10

O. annularis -

O. faveolata, 2021

10 (2 L) 6 substrates per

container, 1 per coating

0.1 T1 to T6 10 C, UNC 30

Abbreviations: Conditioned (C), and Unconditioned (UNC).

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

validations revealed that there was no over-

dispersion. Likelihood ratio chi-square (LR

Chisq) was performed for statistical infer-

ence. Tukey tests for post hoc analysis were

then used. Kaplan-Meier survival analysis was

performed to determine differences in survi-

vorship between substrate coatings (C1 to C6

and T1 to T6, six levels) in the O. annularis

and O. faveolata choice experiments. Survival

between the substrate coatings (C1 to C6 and

T1 to T6, six levels) was compared using log-

rank tests. All statistical tests were carried out

using R (R Core Team, 2019), and the results

were plotted using the ggplot2 package (Wick-

ham, 2009). Data are presented as mean ±

standard error (SE).

RESULTS

Coatings with combinations of properties

For Orbicella annularis a maximum of

258 settlements was recorded on the coatings,

representing 4.30 % of the 6 000 larvae placed

in the containers. A maximum of 171 O. faveo-

lata settlers was recorded on the substrate coat-

ings, representing 2.85 % of the 6 000 larvae

used in the experiment.

Choice experiment: Despite the low set-

tlement, distinguishable differences were found

in the number of O. annularis settled larvae

(Fig. 1) (LR Chisq = 25.74, P = 0.0001), with

a greater number of settlers found on substrate

C1 when compared to substrate C4 (Tukey, P =

0.0015), and C6 (Tukey, P = 0.02). The number

of settlers was significantly greater on substrate

C2 when compared to substrate C4 (Tukey, P

= 0.007). A total of 196 (3.3 ± 0.43) settlers

were observed on the coatings of the substrates

on day eight after spawning, 133 (2.0 ± 0.25)

on day 13, and 58 (0.9 ± 0.18) on day 18. This

shows that the highest number of settlements

for this experiment occurred between day three

and day 12 after spawning under these condi-

tions. Similarly, for O. faveolata significant

differences were found in the number of settled

larvae (Fig. 1) (LR Chisq = 12.27, P = 0.03),

with lower settlement on substrate C4 when

compared to substrate C2 (Tukey, P = 0.04). A

total of 27 (0.4 ± 0.10) settlers were observed

on the upper surface of substrates on day eight

after spawning, 139 (2.2 ± 0.27) on day 13, and

109 (1.8 ± 0.27) on day 18. This shows that the

highest number of settlements for this experi-

ment occurred between days nine and 13 after

spawning under these conditions.

Post-settlement survival: The highest

number of polyps settled was on substrates

C1 and C2 compared to other substrates. The

Kaplan-Meier survival curves demonstrated a

mean survival time of 14.00 ± 0.68 days after

settlement on substrate C1, 13.37 ± 0.37 days

on substrate C2, 13.00 ± 0.00 days on substrate

C3, 16.89 ± 1.23 days on substrate C5 and 18.6

± 4.08 days on substrate C6 (Fig. 2A). The sur-

vival time was significantly lower in substrate

C2 than substrate C5 (Log-rank test, P = 0.02).

A total of 109 O. faveolata primary polyps

were transferred to aquaria after the last count,

distributed between the substrate types, mostly

on C1 and C2 substrates. The Kaplan-Meier

survival curves revealed a mean survival time

of 13.00 ± 0.00 days after settlement on sub-

strate C1, C4, and C5, 14.17 ± 0.68 days on

Fig. 1. The maximum number of O. annularis, and O.

faveolata larvae settled on substrates with coatings with

combinations of properties. Values are means (N = 10), and

error bars represent ± standard error.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

substrate C2, 13.74 ± 0.74 days on substrate

C3 and 17.00 ± 2.17 days on substrate C6 (Fig.

2B). No significant differences were found in

the survival time between substrate coatings

(Log-rank test, P = 0.08).

Coatings with a single property

For O. annularis, a maximum of 64 settlers

was recorded on the substrate coatings, repre-

senting 3.20 % of the 2 000 larvae originally

placed in all containers. A maximum of 33 O.

faveolata settlers was recorded on the substrate

coatings, representing 1.65 % of the 2 000 lar-

vae placed in the containers.

Choice experiment: Although the settle-

ment was low, differences were found in the

number of O. annularis settled larvae between

the coatings (Fig. 3) (LR Chisq = 18.96, P =

0.002), with a significantly greater number of

settlers found on substrate T6 when compared

to substrate T2 (Tukey, P = 0.03), and T4

(Tukey, P = 0.046). No significant difference

was found between substrate conditioning and

lack of conditioning (Fig. 4) (LR Chisq = 0.63,

Fig. 2. Survival probability of primary polyps on substrates with coatings with combinations of properties (C1 to C6) for 41

days after settlement (DAS). A. O. annularis. B. O. faveolata.

Fig. 3. The maximum number of O. annularis and O.

faveolata larvae settled on substrates with coatings with a

single property. Values are means (N = 10), and error bars

represent standard error.

Fig. 4. The maximum number of O. annularis, and O.

faveolata larvae settled on conditioned and unconditioned

substrates. Coatings with a single property (T1 to T6).

Values are means (N = 30), and error bars represent

standard error.

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

P = 0.43). A total of 16 (0.27 ± 0.09) settlers

were observed on the coatings of the substrates

on day eight after spawning, 38 (0.63 ± 0.15)

on day 13, and 11 (0.18 ± 0.07) on day 18. This

shows that the highest number of settlers for

this experiment occurred between day nine and

day 13 after spawning under these conditions.

For O. faveolata no significant difference

was found in the number of settled larvae

between the substrate coatings (Fig. 3) (LR

Chisq = 1.90, P > 0.05). However, differences

were found between substrate conditioning

(Fig. 4) (LR Chisq = 13.92, P = 0.0002),

with a significantly greater number of settlers

found on conditioned substrates compared to

unconditioned substrates. A total of 17 (0.28 ±

0.11) settlers were observed on the coatings of

the substrates on day eight after spawning, 21

(0.35 ± 0.11) on day 13, and 9 (0.15 ± 0.07) on

day 18. This shows that the highest number of

settlers for this experiment occurred between

days nine and 13 after spawning under these

conditions.

Post-settlement survival: A total of 11 O.

annularis primary polyps were transferred to

aquaria after the last count, distributed between

T1, T3, T5, and T6 substrate types, with the

highest number of polyps settled on substrates

T1. The Kaplan-Meier survival curves dem-

onstrated a mean survival time of 7.00 ± 0.00

days after settlement on substrate T1 and T3,

28.00 ± 0.00 days on substrate T5 and 17.50

± 3.50 days on substrate T6 (Fig. 5A). No sig-

nificant differences were found in the survival

time between substrate coatings (Log-rank

test, P > 0.05).

Nine O. faveolata primary polyps were

transferred to aquaria after the last count,

distributed between T1, T5, and T6 substrate

types, mostly on T6 substrates. The Kaplan-

Meier survival curves revealed a mean survival

time of 7.00 ± 0.00 days after settlement on

substrate T1, 31.50 ± 10.5 days on substrate

T5, and 14.00 ± 2.21 days on substrate T6 (Fig.

5B). No significant differences were found in

the survival time between substrate coatings

(Log-rank test, P > 0.05).

DISCUSSION

Overall, our results show that the presenta-

tion of substrates with distinct chemical coat-

ings and combinations to two species of coral

larvae results in low but distinct settlement

patterns and post-settlement survival.

For O. annularis, the substrate with the

combination of properties with a high level

of hydrophobicity combined with light blue

phosphorescent micro-particles (C1) and the

substrate with a high level of hydrophobicity

and red-orange phosphorescent micro-particles

(C2) were the preferred settlement substrates

when compared to the other combinations.

Fig. 5. Survival probability of primary polyps on substrates with coatings with a single property (T1 to T6) throughout 42

days after settlement (DAS). A. O. annularis. B. O. faveolata.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

Nevertheless, the experiment with a single

property revealed that the ceramic control (T6)

was preferred over intermediate hydrophobic-

ity (T4) and light blue phosphorescent micro-

particles (T2). For O. faveolata, we found that

a high level of hydrophobicity, combined with

the red-orange (C2) phosphorescent micro-

particles, was the preferred combination as

a settlement substrate when compared to the

substrate with intermediate hydrophobicity and

hydroxyapatite coating (C4). This means that

combinations of properties generate successful

settlement on these substrates. Individual prop-

erties did not generate this effect. The fact that

the greatest number of settlers was observed on

substrates with phosphorescent microparticles,

and a high level of hydrophobicity confirms

that these species use photosensitivity and

respond to colour, in this case, light blue and

red-orange, to fine-tune the selection of appro-

priate substrates or microhabitats (Mason et al.,

2011). This photosensitivity towards certain

wavelengths of light may assist larvae in select-

ing a microhabitat (Strader et al., 2015), serv-

ing as a form of depth gauge (Mason & Cohen,

2012) or guide, given that the distribution of

light on coral reefs is highly heterogeneous at

the microhabitat level due to depth, water clar-

ity, bottom slope, substrate type and exposure

(Brakel, 1979).

The substrate types determined the sur-

vival rates of O. annularis and O. faveolata

recruits. On transferring the recruits to aerated

aquaria, the number of polyps that survived on

the substrates dropped abruptly, especially for

those coated with polyester resin. This was evi-

denced by the fact that in the experiment with

coatings with combinations of properties, the

longest survival time (41 days after settlement)

was associated with the only substrate without

a polyester resin coating (C6). In the experi-

ment with coatings with a single property, we

observed the same pattern; the longest survival

time (42 days after settlement) was on a sub-

strate without this polyester resin coating (T5).

The polyester resin used is somewhat soluble

in water; hence the resin may have slowly

dissolved, resulting in an unstable substrate,

possibly preventing viable adhesion and com-

promising long-term post-settlement survival.

Additionally, hydrophobic surfaces generate a

contact angle with the water greater than 90°.

When this contact angle is greater, the adhe-

sion force to this surface will be less (Lejars et

al., 2012). Therefore, hydrophobicity generates

a weaker adhesion force between the recruit

and the substrate and possibly, when generat-

ing movement in the aquarium system or due

to aeration, the settlers detached from the

substrate possibly due to a weak attachment.

Therefore, using these hydrophobic surfaces

to prevent the colonization of other organ-

isms, such as filamentous algae, can also be

detrimental to coral survival since they func-

tion as antifouling coatings that allow for easy

release when water motion is applied (Lejars et

al., 2012). This would mean that, even though

the combination of high hydrophobicity and

phosphorescent micro-particles presents the

most favourable settlement option compared

to other combinations, the component used to

create intermediate hydrophobicity (polyester

resin) and high hydrophobicity may have been

detrimental to settler attachment.

However, even though substrate condition-

ing is known to encourage settlement (Erwin

et al., 2008), in the experiment with coatings

with combinations of properties, it should also

be noted that none of the substrates used in

the O. annularis and O. faveolata experiments

were conditioned; hence no biofilm could have

formed at their surfaces, yet larvae settled

successfully, which means that conditioning

is unnecessary for these substrates. This repre-

sents an advantage over other substrates (Har-

rison & Wallace, 1990; Patterson et al., 2016;

Petersen et al., 2005) that require conditioning

prior to larval settlement. However, for the

experiment with coatings with a single prop-

erty, we found that the settlement of O. faveo-

lata was greater on conditioned substrates; this

may indicate that these substrates with a single

property are not attractive enough on their own

to larvae and require conditioning to generate

an adequate settlement cue for coral larvae.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71 (S1): e54864, abril 2023 (Publicado Abr. 30, 2023)

Orbicella annularis and O. faveolata lar-

vae showed different behaviours, most notably

in their settlement patterns. For O. faveolata,

most larvae settle within the first two weeks

if an appropriate site is found, with 80 % of

larvae remaining in the plankton for more than

eight days before attempting to settle (Szmant

& Meadows, 2006). Our results show that the

peak settlement period for O. faveolata larvae

was between nine and 13 days after spawn-

ing. For O. annularis, the settlement peak was

between three and eight days after spawning.

This behaviour may also affect the affinity

that O. annularis larvae have for the substrates

with the combination of properties. More O.

annularis larvae settled and they settled more

rapidly when compared to O. faveolata larvae.

Furthermore, we did not find this pattern in O.

annularis in the single-property experiment,

possibly because the larvae were not attracted

to the substrates. Thus, they took more time to

settle and were therefore settled in a time frame

similar to O. faveolata larvae.

In conclusion, this is the first study to

report the ability of O. annularis and O.

faveolata to detect phosphorescence and hydro-

phobicity during the substrate selection pro-

cess, which could be key to micro-habitat

selection. Furthermore, this is the first time

that the effects of substrate surface properties

have been tested on these species, with vary-

ing effects. On the one hand, microparticles

with blue and red-orange phosphorescence and

high hydrophobicity favour settlement in O.

annularis and O. faveolata, but some of the

components used to coat the substrates may be

detrimental to the survival of the polyps over

time. These issues need to be addressed as

long-term survival is critical in restoration proj-

ects involving the outplanting of coral settlers.

Finally, it is essential to keep testing different

combinations of substrate surface properties to

determine the ones that are most conducive to

larval settlement and polyp survival as well as

being the least favourable to fleshy algal settle-

ment. This will help us to identify the most

suitable substrates for each species and is vital

for optimizing future restoration efforts.

Ethical statement: the authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGEMENTS

We wish to thank members of the Cora-

lium laboratory for sharing their knowledge

and support in the field and the lab, particularly

Gandhi Ramírez, Raul Tecalco, and Sandra

Mendoza. Also, to Pablo Andrés Guzmán for

his help with statistical analyses and expertise

in R Software.

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