Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58880, marzo 2024 (Publicado Mar. 31, 2024)

Recent molecular techniques to strengthen

ecological studies in echinoderms

Ruber Rodríguez-Barreras *1; http://orcid.org/0000-0001-7790-6108

1. Department of Biology, University of Puerto Rico, Mayagüez campus; PO Box 9000, Mayagüez, Puerto Rico; ruber.

rodriguez@upr.edu (*Correspondence)

Received 04-VI-2023. Corrected 07-IX-2023. Accepted 25-XI-2023.

ABSTRACT

Introduction: Echinoderms, an integral component of marine ecosystems worldwide, have captivated sci-

entific interest for centuries. Despite this longstanding attention, comprehending key facets such as trophic

relationships, diet composition, and host-microbiota relationships still represents a challenge using traditional

techniques. Recent years, however, have witnessed a transformative shift, thanks to the emergence of advanced

molecular techniques, offering new approaches to strengthen ecological studies in echinoderms.

Objective: Explore how recent advancements in molecular tools have impacted ecological research on echino-

derms. Specifically, we aim to investigate the potential of these tools to shed light on trophic interactions, diet

composition, and the characterization of gut microbial communities in these organisms.

Methods: Available literature was used to clarify how novel molecular techniques can improve ecological studies.

The focus is diet, trophic relationships, and gut microbiota.

Results: Traditionally, studies of stomach contents using compound microscopy have provided an idea of ingest-

ed material; nevertheless, sometimes a simple magnified visualization of dietary content does not allow exhaus-

tive identification of the entire food spectrum, as it is limited due to the rapid digestion and maceration of food

items within the echinoderm’s digestive tract. The use of DNA-metabarcoding, targeting specific DNA regions,

such as the mitochondrial COI gene, has allowed us to enhance the accuracy and precision of diet characteriza-

tion by enabling the identification of prey items down to the species or even genetic variant level, providing valu-

able insights into specific dietary preferences. Another approach is the use of stable isotopes, particularly carbon

and nitrogen, which provide a powerful tool to trace the origin and flow of nutrients through food webs. By

analyzing the isotopic signatures in muscular tissues and food items, we can discern the sources of their primary

food items and gain insights into their trophic position within the ecosystem. Lastly, a third new technique used

to elucidate the characterization of the prokaryotic community is 16S rRNA sequencing. This method allows us

to explore the composition and dynamics of the digestive tract microbial communities.

Conclusions: This is a promising era for ecological research on echinoderms, where advances of molecular tools

have enabled an unprecedented level of detail, resolving longstanding challenges in comprehending their trophic

interactions, diet composition, and host-microbiota relationships, and opening new avenues of investigation in

ecological studies.

Key words: echinoderms; stable isotopes; 16S rRNA; DNA-metabarcoding; ecology; review.

RESUMEN

Técnicas moleculares recientes que fortalecen los estudios ecológicos de equinodermos

Introducción: Los equinodermos, un componente integral de los ecosistemas marinos en todo el mundo, han

captado el interés científico durante siglos. A pesar de esta prolongada atención, el comprender facetas clave

https://doi.org/10.15517/rev.biol.trop..v72iS1.58880

SUPPLEMENT

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58880, marzo 2024 (Publicado Mar. 31, 2024)

INTRODUCTION

The use of novel technologies in eco-

logical studies on echinoderms and other

marine organisms has significantly advanced

our understanding of ecology, physiology, and

interactions within marine ecosystems. Among

these techniques, stable isotope analysis pro-

vides valuable insights into trophic relationships

and nutrient flow within marine ecosystems

(Phillips & Gregg, 2003). The use of stable iso-

topes in ecological studies has its origins in the

field of geochemistry, where isotopic analysis

was initially used to understand the composi-

tion and age of rocks and minerals (O’Connell

et al., 2012). The application of stable isotopes

in ecology began in the 1960s when researchers

realized that isotopic signatures could be used

to trace the movement of elements through

ecosystems, using stable carbon isotopes to

study the carbon cycle and its impact on climate

change (Keeling et al., 1995). Since then, stable

isotopes have become valuable tools in the

study of ecological processes, such as trophic

interactions, nutrient cycling, and migration

patterns. The development of new analytical

techniques and advancements in mass spec-

trometry have further enhanced the application

of stable isotopes in the field of ecology. The

Isotope ratio mass spectrometry (IRMS) meth-

od allows researchers to measure the ratios of

isotopes in a sample with high precision and

accuracy (Godin & McCullagh, 2011; Hayes,

2001). The development of compound-specific

isotope analysis (CSIA) has allowed researchers

to examine isotopic signatures at the molecular

level, providing more detailed insights into

ecological processes (Chikaraishi et al., 2007).

These advancements have opened new avenues

for studying ecological dynamics and have

como las relaciones tróficas, la composición de la dieta y las relaciones huésped-microbiota todavía representa un

desafío utilizando técnicas tradicionales. Sin embargo, los últimos años han sido testigos de un cambio transfor-

mador, gracias a la aparición de técnicas moleculares avanzadas, que ofrecen nuevos enfoques para fortalecer los

estudios ecológicos en equinodermos.

Objetivo: Explorar cómo los avances recientes en herramientas moleculares han impactado la investigación

ecológica sobre equinodermos. Específicamente, nuestro objetivo es investigar el potencial de estas herramientas

para arrojar luz sobre las interacciones tróficas, la composición de la dieta y la caracterización de las comunidades

microbianas intestinales en estos organismos.

Métodos: Se utilizó la literatura disponible para aclarar cómo las nuevas técnicas moleculares pueden mejorar los

estudios ecológicos. La atención se centra en la dieta, las relaciones tróficas y la microbiota intestinal.

Resultados: Tradicionalmente, los estudios del contenido estomacal mediante microscopía compuesta han pro-

porcionado una idea del material ingerido; Sin embargo, a veces una simple visualización ampliada del contenido

dietético no permite una identificación exhaustiva de todo el espectro alimentario, ya que está limitado debido a

la rápida digestión y maceración de los alimentos dentro del tracto digestivo del equinodermo. El uso de meta-

barcoding de ADN, dirigidos a regiones específicas del ADN, como el gen COI mitocondrial, nos ha permitido

mejorar la exactitud y precisión de la caracterización de la dieta al permitir la identificación de presas hasta el

nivel de especie o incluso de variante genética, lo que proporciona valiosos resultados sobre preferencias dietéticas

específicas. Otro enfoque es el uso de isótopos estables, en particular carbono y nitrógeno, que proporcionan una

poderosa herramienta para rastrear el origen y el flujo de nutrientes a través de las redes alimentarias. Al analizar

las firmas isotópicas en los tejidos musculares y los alimentos, podemos discernir las fuentes de sus alimentos

primarios y obtener información sobre su posición trófica dentro del ecosistema. Por último, una tercera técnica

nueva utilizada para dilucidar la caracterización de la comunidad procariótica es la secuenciación del ARNr

16S. Este método nos permite explorar la composición y dinámica de las comunidades microbianas del tracto

digestivo.

Conclusiones: Esta es una era prometedora para la investigación ecológica sobre equinodermos, donde los

avances de las herramientas moleculares han permitido un nivel de detalle sin precedentes, resolviendo desafíos

de larga data en la comprensión de sus interacciones tróficas, composición de la dieta y relaciones huésped-

microbiota, y abriendo nuevas vías de investigación. en estudios ecológicos.

Palabras clave: equinodermos; isótopos estables; ARNr 16S; metabarcoding de ADN; ecología; revisión.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58880, marzo 2024 (Publicado Mar. 31, 2024)

contributed significantly to our understanding

of complex ecological systems.”

Another powerful technique that has sig-

nificantly advanced ecological studies involv-

ing echinoderms is the application of DNA

sequencing methods for prokaryotic and

eukaryotic organisms. This molecular tool

allows researchers to explore the diversity and

composition of microbial communities associ-

ated with echinoderms (Leray & Knowlton,

2016; Silva et al., 2021). The use of 16S rRNA in

ecological studies has its origins in genetics and

molecular biology. 16S rRNA is a ribosomal

RNA sequence present in the DNA of prokary-

otic and some eukaryotic cells, which encodes

for structural components of the ribosome. In

the 1980s, the technique of 16S rRNA sequenc-

ing was first used to investigate the diversity

and phylogenetic relationships among different

bacterial species (Oren, 2004; Whitman et al.,

2022). This approach revolutionized the field

of microbial ecology by providing a molecular

tool to identify and classify microorganisms

based on their genetic relatedness. By compar-

ing the sequences of 16S rRNA genes from dif-

ferent organisms, researchers can reconstruct

the evolutionary history of bacteria and infer

their ecological roles in various ecosystems.

Advancements in DNA sequencing technolo-

gies have further propelled the use of 16S

rRNA in ecological studies. The development

of high-throughput sequencing platforms, such

as next-generation sequencing, has enabled

researchers to analyze large volumes of 16S

rRNA sequences from environmental samples

in a cost-effective and time-efficient manner

(Caporaso et al., 2010; Muhamad-Rizal et al.,

2020). This has facilitated the exploration of

microbial diversity and community composi-

tion in several ecological settings, ranging from

soil and marine environments to the human

gut microbiome. Additionally, bioinformatics

tools and databases specifically designed for

16S rRNA analysis, such as QIIME (Quanti-

tative Insights Into Microbial Ecology) and

Greengenes, have emerged, providing research-

ers with computational resources to process

and interpret vast amounts of sequencing data

(DeSantis et al., 2006; Gilbert et al., 2014).

DNA metabarcoding, is a third novel tech-

nique that has emerged as a transformative tool

for ecological studies in echinoderms. This

technique allows for high-throughput identi-

fication of species based on DNA sequences

extracted from environmental samples (Leray

& Knowlton, 2015). By targeting specific DNA

regions, such as the mitochondrial COI gene,

researchers can rapidly assess echinoderm

diversity, community structure, and species

interactions in various marine habitats (Leray

& Knowlton, 2016; Leray et al., 2013). DNA

metabarcoding involves the amplification and

sequencing of specific DNA regions, such as the

barcode regions, to identify and quantify the

diversity of organisms present in environmen-

tal samples. The concept of DNA barcoding

was initially proposed to identify species based

on short, standardized DNA sequences (Antil

et al., 2023; Hebert et al., 2003). However, it

was soon realized that this approach could be

extended to ecological studies by simultane-

ously sequencing multiple DNA samples and

generating large datasets for species identifica-

tion and community analysis. The develop-

ment of next-generation sequencing platforms,

such as Illumina sequencing, provided the

technological capacity to process a massive

number of DNA sequences at a reasonable

cost, making DNA metabarcoding a powerful

tool in ecological research (Hassan et al., 2022;

Pawlowski et al., 2022). By targeting differ-

ent barcode regions, DNA metabarcoding can

provide a comprehensive and high-resolution

assessment of biological communities. Fur-

thermore, it can be applied to other organ-

isms, including plants, animals, prokaryotes,

and microeukaryotes by selecting appropriate

barcode regions. The interpretation of DNA

metabarcoding data relies on reference data-

bases that contain known DNA sequences from

a wide range of organisms. These databases,

such as GenBank and the Barcode of Life Data

Systems (BOLD), allow for the identification

and taxonomic assignment of the sequenced

DNA fragments (Taberlet et al., 2012). With

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advances in bioinformatics tools and analytical

pipelines designed specifically for DNA metab-

arcoding analysis, researchers can now extract

valuable ecological information from complex

and diverse environmental samples (Hassan et

al., 2022; Zhang et al., 2023).

These novel techniques and their multiple

variations have important implications for con-

servation and ecosystem management, con-

tributing to our understanding of echinoderm

species and their roles within marine ecosys-

tems. Therefore, the main goal of this review

is to analyze the fast advance of new molecular

tools that allows a level of detail never seen

before, clarifying old doubts, and opening new

research avenues in ecological studies.

Stable isotopes

The complexity of trophic relationships

exceeds common perception. Simply exam-

ining the contents of an organism’s stomach

does not reveal which specific items are being

absorbed by the consumer (Peterson & Fry,

1987; Phillips et al., 2014). Stable isotopes

have emerged as a valuable tool for studying

the ecology and physiology of echinoderms, a

marine group of invertebrates that includes sea

lilies, brittle stars, starfish, sea urchins, and sea

cucumbers, providing insights into the trophic

interactions, migration patterns, and habitat

preferences of echinoderms (Fry & Sherr, 1984;

Sturbois et al., 2022). Carbon, nitrogen, oxygen,

and sulfur isotopes are commonly employed

to unravel several aspects of echinoderm biol-

ogy (Cabanillas-Terán et al., 2016; Rodríguez-

Barreras et al., 2015; Wangensteen et al., 2011).

In recent years, stable isotope analysis has been

extensively used to examine the trophic ecology

of echinoderms, shedding light on their feed-

ing preferences, diet breadth, and interactions

with other organisms (Cabanillas-Terán et al.,

2019; Pérez-Posada et al., 2023). For instance,

Rodríguez-Barreras et al. (2016) presented the

ellipses-based metrics of niche width for five

Caribbean sea urchins based on habitat prefer-

ences where each species displayed a particu-

lar isotopic signature of carbon and nitrogen

indicating no food overlapping among them,

despite of species from the same habitat can be

found inhabiting close to each other (Fig. 1).

Among stable isotopes, Carbon, and par-

ticularly carbon-13 (δ13C), play a crucial role

in understanding the feeding ecology of echi-

noderms (Purcell et al., 2007). By analyzing

the carbon isotopic signatures in muscular

tissues, researchers can decipher the primary

carbon sources they consume, providing valu-

able information about their dietary prefer-

ences and trophic positions within marine food

webs (Burton et al., 2011; Carney, 2010; Gale

et al., 2013). Moreover, nitrogen isotopes, such

as nitrogen-15 (δ15N), have proven instrumen-

tal in studying the trophic levels and nutrient

sources (Hobson et al., 1995; Nilsen et al.,

2008). The δ15N values in their tissues increase

with higher trophic positions, offering insights

into their position in the food chain and poten-

tial dietary shifts (Gurney et al., 2001). Fur-

thermore, stable nitrogen isotopes have been

also used to explore the effects of anthropo-

genic nutrient inputs on echinoderm commu-

nities and to assess the ecological impacts of

Fig. 1. Results of the ellipses-based metrics of niche width

for two echinoids groups based on habitat preferences

where circles represent the potential niche of each species.

A. Represents three species from reefs biotopes, and B.

represents two species from seagrass beds.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58880, marzo 2024 (Publicado Mar. 31, 2024)

eutrophication (Radabaugh et al., 2013; Yatsuya

& Nakahara, 2004). The ratios of 13C to 12C and

15N to 14N in the tissues compared to their algae

food resources is referred to as Trophic Enrich-

ment Factor (Parnell et al., 2010). Thus, we can

also calculate the trophic level for every indi-

vidual, using the equation proposed by Hobson

and Welch (1992).

Additionally, oxygen isotopes, such as oxy-

gen-18 (δ18O), are employed to determine the

thermal history and evolutionary patterns of

dispersal (Gerringer 2019; Zenteno et al., 2013).

Changes in δ18O values in skeletal structures

can indicate temperature changes or move-

ments across different water conditions (Kill-

ingley & Rex, 1985). This isotope also provides

insights into the thermal ecology and habi-

tat preferences, helping to understand their

responses to climate change and oceanographic

processes (Luo et al., 2023; Tiwari et al., 2013;

Zhao et al., 2020). The fourth most common

used stable isotope are sulfur isotopes, particu-

larly sulfur-34 (δ34S), contribute to understand-

ing the nutrient sources and sulfur cycling in

food webs (Connolly et al., 2004). The δ34S

values in their tissues can reflect the use of dif-

ferent sulfur sources, such as marine organic

matter or sulfide-rich sediments (Vaslet et al.,

2012), but also, researchers can gain insights

into the influence of the prokaryotic commu-

nity in the diet of echinoderms (Mendes et al.,

1963; Pascal et al., 2017).

Stable isotopes have also proven valuable

for studying the physiology and metabolic pro-

cesses of echinoderms. For example, carbon

isotopes have been used to investigate metabolic

rates and energy allocation in these organisms

(Sun et al., 2012). By tracing the input of isoto-

pically labeled compounds, obtaining insights

into nutrient uptake, assimilation, and utiliza-

tion (Walters et al., 2021). Thus, stable isotope

analysis has also been employed to study the

reproductive biology and larval ecology of echi-

noderms. Nitrogen isotopes have been used to

trace the transfer of maternal nutrients to devel-

oping embryos and larvae (Reitzel & Miner,

2007). By analyzing the isotopic composition of

different life stages, marine ecologist can gain

insights into larval dispersal patterns, connec-

tivity between populations, and the influence of

local versus distant sources of larvae (Lester et

al., 2007; Levin, 2006). Lastly, the combination

of stable isotope analysis with other techniques,

such as fatty acid analysis and genetic markers,

has provided a comprehensive understanding

of the feeding ecology and population structure

in echinoderms (McKenzie et al., 2000; North

et al., 2019). These integrated approaches allow

researchers to examine the dietary preferences

of different echinoderm species, identify poten-

tial competition among species, and investigate

the drivers of population dynamics (Howell et

al., 2003; Rossi & Elias-Piera, 2018).

Eukaryotic DNA-metabarcoding

This method provides a comprehensive

and efficient approach to studying the biodi-

versity and ecological patterns of echinoderms,

aiding in conservation efforts, and inform-

ing ecosystem management strategies (Leray

& Knowlton, 2015; Leray et al., 2013; Ric-

cioni et al., 2022). DNA metabarcoding, a

high-throughput sequencing technique, has

revolutionized the field of biodiversity assess-

ment and ecological studies (Barnes & Turn-

er, 2016). This powerful tool allows for the

rapid identification of species and the analysis

of complex biological communities based on

DNA markers. In recent years, DNA metaba-

rcoding has emerged as a valuable approach

in echinoderm research, enabling scientists

to gain insights into the diversity, community

structure, and ecological roles of these marine

organisms (Licuanan & Matias, 2022; Okanishi

et al., 2023; Rodriguez-Barreras et al., 2020).

This essay aims to explore the applications

of DNA metabarcoding in echinoderm stud-

ies, highlighting its contributions to taxonomy,

community ecology, trophic interactions, and

conservation. In addition, DNA metabarcoding

has proven to be a reliable method for species

identification and taxonomy in echinoderms.

By sequencing specific gene regions, such as the

mitochondrial cytochrome c oxidase subunit I

(COI) gene, researchers can accurately identify

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and differentiate echinoderm species, includ-

ing cryptic and morphologically similar taxa

(Wangensteen et al., 2018). This non-invasive

approach overcomes the limitations of tradi-

tional morphological identification and has led

to the discovery of new species and the revision

of taxonomic classifications in echinoderms

(Borrero-Pérez et al., 2019; Trivedi et al., 2016).

DNA metabarcoding has also been instru-

mental in elucidating the community struc-

ture and diversity of echinoderm assemblages.

By analyzing environmental DNA (eDNA)

extracted from seawater or sediment samples,

researchers can assess the presence and abun-

dance of different echinoderm species in each

habitat (Deiner et al., 2016; Leray et al., 2013).

This approach provides a comprehensive and

efficient method for studying echinoderm

biodiversity, particularly in hard-to-access or

cryptic habitats and allows for the monitor-

ing of community dynamics and changes over

time (Boissin et al., 2017; Deiner et al., 2016).

Moreover, DNA metabarcoding enables the

identification of rare or endangered species,

contributing to their conservation and manage-

ment. On the other hand, DNA metabarcoding

has shed light on the trophic interactions and

feeding ecology of echinoderms. By analyzing

the gut contents or fecal material of echino-

derms, researchers can identify the prey items

consumed by these organisms and investigate

their dietary preferences and ecological roles

(Jia et al., 2022; Rodríguez-Barreras et al.,

2020). This information is crucial for under-

standing energy flow and nutrient cycling in

marine ecosystems and can contribute to the

assessment of ecosystem health and function-

ing (Bourlat et al., 2013; Compson et al., 2018).

Likewise, DNA metabarcoding can reveal the

occurrence of symbiotic or commensal organ-

isms associated with echinoderms, providing

insights into their ecological interactions and

mutualistic relationships (Kodama et al., 2023;

Toju, 2015).

DNA metabarcoding has proven particu-

larly valuable in studying the trophic ecology

of echinoderms, shedding light on the trophic

interactions and food web dynamics involving

echinoderms (Baure et al., 2023; Redd et al.,

2014). Thus, a recent study described the food

items found in four Caribbean sea urchins

(Rodríguez-Barreras et al., 2020). Authors also

compared diet similarities among species from

the same habitat (Fig. 2). Through the analy-

sis of gut contents from 60 individuals across

three collection sites, we found that these four

sea urchins primarily feed on seaweeds, with

additional consumption of small protists, fungi,

and metazoans. Notably, variations in diet were

observed both between species and among col-

lection sites, suggesting potential inter-specific

competition and niche overlap among these

generalist omnivores. Hence, genetic studies

have been performed in echinoderms (Alcudia-

Catalma et al., 2020; Layton et al., 2016). For

example, the study of 19 sea cucumber species,

finding low genetic variation within most spe-

cies (Alcudia-Catalma et al., 2020). Another

study analyzed the effectiveness of DNA bar-

coding for species identification in a diverse

range of marine invertebrates, including crusta-

ceans, mollusks, polychaetas, and echinoderms.

By analyzing nearly half of the 300 known

Echinodermata species in Canadian waters,

the research highlighted six species requiring

further taxonomic investigation due to their

specimens falling into two or three distinct

sequence clusters. The study also explored the

potential impacts of larval dispersal and glacial

events on genetic diversity patterns in 19 trans-

oceanic species (Layton et al., 2016).

Prokaryotic 16S metabarcoding

Traditional methods used in microbiol-

ogy, such as culture and microscopy, have

provided evidence of the presence of bacte-

ria within the gut microbiota of sea urchins.

Many of these bacteria are associated with

ecological interactions and metabolic processes

(Marangon et al., 2023; McCracken et al., 2023;

Temara et al., 1993). However, recent advance-

ments in molecular sequencing techniques have

emerged as powerful tools that are revolution-

izing the characterization of microbiota in

marine organisms (Hakim et al., 2016; Nelson

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58880, marzo 2024 (Publicado Mar. 31, 2024)

et al., 2010; Petti et al., 2005; Rodríguez-Bar-

reras et al., 2021). These molecular approaches

have significantly enhanced our understanding

of the microbial communities associated with

echinoderms and other marine invertebrates,

enabling a more comprehensive analysis of

their composition and function.

The 16S ribosomal RNA (16S rRNA),

present in all bacteria and archaea, serves as

a molecular marker to identify and classify

microbial taxa (Hakim et al., 2019). By analyz-

ing the 16S rRNA gene sequences, researchers

can unravel the intricate relationships between

echinoderms and their associated microbial

communities, shedding light on the potential

roles of these microbes in echinoderm health,

nutrition, and overall ecological functioning

(Lima-Mendez et al., 2015; Rodríguez-Barreras

et al., 2023). The 16S rRNA gene is a widely

used molecular marker for studying microbial

diversity and community structure. In recent

years, the application of this marker sequenc-

ing has expanded to include investigations

of microbial associations in various ecosys-

tems, including the marine environment. This

essay explores the applications of 16S rRNA

Fig. 2. A nMDS ordination (Bray–Curtis) for COI samples of four sea urchins Diadema antillarum (Philippi, 1845),

Echinometra lucunter (Linnaeus, 1758), Lytechinus variegatus (Lamarck, 1816) and Tripneustes ventricosus (Lamarck, 1816),

collected from three sites of Puerto Rico. Circles, triangles, and squares identify individuals of each species. In each case,

individuals of the same species are grouped together.

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sequencing in echinoderm research, highlight-

ing its contributions to understanding the

microbial interactions, symbiosis, and envi-

ronmental adaptations.

The use of 16S rRNA sequencing has

revealed valuable insights into the microbial

communities associated with echinoderms. By

analyzing the V4 region of the 16S rRNA

gene, researchers have identified diverse bac-

terial taxa that inhabit the surfaces, tissues,

and gut of echinoderms (Jackson et al., 2018).

This approach has enabled the characterization

of core microbiomes in echinoderms across

different species, providing a foundation for

understanding the functional roles of microor-

ganisms in echinoderm health, development,

and physiology. In addition, the use of 16S

rRNA sequencing has provided insights into the

microbial contributions to echinoderm physiol-

ogy and environmental adaptations. Studies

have identified specific bacterial taxa associ-

ated with nutrient metabolism, detoxification

processes, and resistance to environmental

stressors in echinoderms (Franzenburg et al.,

2012; Marangon et al., 2021). This knowledge

enhances our understanding of the functional

capabilities of echinoderm-associated micro-

organisms and their potential roles in host

health, resilience, and acclimation to changing

environmental conditions.

Furthermore, through 16S rRNA sequenc-

ing, researchers can decipher intriguing micro-

bial interactions within echinoderms. For

instance, recent studies revealed the occurrence

of potential symbiotic relationships between

echinoderms and specific bacterial taxa, sug-

gesting mutualistic or commensal associations

(Brigmon & De Ridder, 1998; Schuh et al.,

2020). These bacterial partnerships may con-

tribute to nutrient acquisition, defense against

pathogens, and overall host fitness. Under-

standing the dynamics and functional roles of

these symbiotic interactions can provide valu-

able insights into the ecology and evolution of

echinoderms (Carrier et al., 2021). In addition

to symbiotic interactions, 16S rRNA sequencing

has provided insights into the dynamic nature

of the microbiome in echinoderms. Studies

have revealed variations in the microbial com-

munity composition across different devel-

opmental stages, habitats, and environmental

conditions (Ketchum et al., 2021; Marangon

et al., 2023; Rodríguez-Barreras et al., 2023).

These findings suggest that the echinoderm

microbiome is influenced by both intrinsic

factors and extrinsic environmental factors,

highlighting the importance of considering the

context-dependent nature of microbial associa-

tions in echinoderm research.

The study of the microbial diversity asso-

ciated with echinoderms using 16S rRNA

sequencing has also provided valuable insights

into host-microbe interactions and disease

dynamics. By examining the microbiomes of

diseased echinoderms, researchers have identi-

fied shifts in microbial composition and poten-

tial pathogens associated with disease symptoms

(Galac et al., 2016; Marangon et al., 2023). This

knowledge contributes to our understanding of

the etiology and progression of diseases affect-

ing echinoderms, enabling the development of

targeted management strategies and conserva-

tion efforts. Another significant application of

16S rRNA sequencing in echinoderm research

is the investigation of microbial-mediated eco-

logical processes, such as nutrient cycling and

organic matter degradation. By characterizing

the functional potential of the echinoderm-

associated microbiota, researchers can gain

insights into the roles of microorganisms in

nutrient transformations, carbon fluxes, and

the overall functioning of marine ecosystems

(Masasa et al., 2023; Thompson & Polz, 2006).

These findings highlight the interconnected-

ness between echinoderms and their associated

microbiomes in ecosystem processes.

The integration of 16S rRNA sequencing

with other omics approaches, such as metage-

nomics and metatranscriptomics, has further

expanded our understanding of the functional

potential and metabolic activities of inverte-

brates-associated microbiomes (Gudenkauf &

Hewson, 2015). These multi-omics approaches

enable the exploration of gene functions, met-

abolic pathways, and ecological interactions

within the microbial communities associated

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e58880, marzo 2024 (Publicado Mar. 31, 2024)

with echinoderms (Rey-Campos et al., 2022).

By combining different molecular techniques,

researchers can unravel the intricate relation-

ships between echinoderms and their microbial

partners, shedding light on the mechanisms

driving their symbiotic associations and ecolog-

ical contributions (Lowe et al., 2017; Voronov et

al., 2023). On the other hand, the use of 16S

rRNA sequencing has significantly advanced

our understanding of the microbiota residing

in the digestive systems. This novel technique.

For instance, recent study revealed the gut

microbiota of sea cucumbers (Pagán-Jiménez et

al., 2019), revealing distinct microbial profiles

among different species of sea cucumbers, and

suggesting the influence of host-specific factors

in shaping the composition of the gut microbio-

ta. Another study examined the gut microbiota

of a sea star and found the existence of specific

microbial taxa involved in nutrient metabolism

and the breakdown of complex organic com-

pounds, highlighting the symbiotic relationship

between the host and its gut microbiota in the

digestive processes of sea stars (McCracken et

al., 2023). In another study conducted with four

Caribbean sea urchins, authors found similar

gut microbiotas among the species, but one of

them (Lytechinus variegatus) displayed specific

microbiota profiles (Rodríguez-Barreras et al.,

2021) (Fig. 3).

16S rRNA sequencing has also revealed

the diversity and dynamics of the digestive

system microbiota in echinoderms (Masasa et

al., 2021; Masasa et al., 2023). For instance, a

recent study confirmed that the microbiome

of algae-eating sea urchins like Tripneustes gra-

tilla (Linnaeus, 1758) plays a significant role in

digesting fiber-rich seaweed, but also the study

revealed unique characteristics in the microbial

communities, particularly in the esophagus and

Fig. 3. Global analyses comparing the four sea urchin species. A. Bray-Curtis analysis represented in a 2D Principal

Coordinates Analysis (PCoA) using species as metadata categories, depicts distinct species clustering with permanova Pvalue=

0.004; Anosim Pvalue< 0.001, B. Rarefaction curves of Chao1 index demonstrated significant differences between green

(Lytechinus_variegatus) and red (Echinometra_lucunter) sea urchin (Pvalue= 0.006) and between red (Echinometra_lucunter)

and black (Diadema_antillarum) sea urchin (Pvalue= 0.048), C. Species relative abundance at phyla, and D. genus levels are

depicted by the bar plots.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e58880, marzo 2024 (Publicado Mar. 31, 2024)

intestine, concluding that the shifting microbial

communities within the digestive tract provide

strong evidence supporting the idea of bacteria

playing a crucial role in food digestion (Masasa

et al., 2023). Furthermore, environmental fac-

tors have been shown to influence the composi-

tion of gut microbiota. A study found that the

relative abundance of certain microbial taxa

with changing temperatures, indicating the

sensitivity of the gut microbiota to environ-

mental variations and the potential impact on

host health and physiology (Dong et al., 2021;

Zhang et al., 2013; Zhang et al., 2019). However,

it is important to remark that these powerful

molecular techniques have some limitations.

For example, the 16S rRNA sequencing only

provides information on Bacteria and some

Archae but fails to detect other members of

the microbiota (fungi, viruses, and unicellu-

lar eukaryotes). This limitation is particularly

striking in the recent publication that a ciliated

eukaryote is the culprit of sea urchin mass mor-

tality (Hewson et al., 2023) a finding that would

not have been possible using 16S technology.

DISCUSSION

The integration of stable isotopes, DNA-

metabarcoding, both eukaryotic and prokary-

otic, has revolutionized ecological studies with

echinoderms, providing valuable insights into

their trophic interactions, biodiversity, and

microbial associations. Stable isotopes have

been instrumental in unraveling the trophic

ecology of echinoderms, shedding light on their

feeding habits, diet preferences, and ecological

roles within marine ecosystems (González-De

Zayas et al., 2020; Hobson, 2023). DNA-metab-

arcoding has transformed the way we assess

echinoderm biodiversity, allowing for efficient

species identification and revealing hidden

diversity and community dynamics (Leray et

al., 2013; Sinniger et al., 2016). Additionally,

16S rRNA sequencing has provided a deeper

understanding of the microbial associations

and functional roles of microorganisms associ-

ated with echinoderms, unraveling the intricate

symbiotic relationships and their ecological

contributions (García-Aljaro et al., 2017; Web-

ster et al., 2019). By leveraging these advanced

techniques, researchers can gain comprehen-

sive insights into the ecology and conserva-

tion of echinoderms. The knowledge obtained

through stable isotopes, DNA-metabarcoding,

and 16S rRNA sequencing contributes to our

understanding of ecosystem functioning, tro-

phic dynamics, and the impacts of environmen-

tal changes on echinoderm populations. This

information is crucial for effective management

and conservation strategies aimed at protecting

these ecologically important organisms and the

marine habitats they inhabit.

Ethical statement: the author declares that

he agrees with this publication; that there is no

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

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgements sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

I would like to express my gratitude to the

organizing committee of the 5th Latin American

Echinoderm Conference, specially to Juan José

Alvarado and Jorge Sonnelholzner, for being

invited to the Congress and presenting the lec-

ture on which this paper is based.

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