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Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(S1): 550-557, March 2021 (Published Mar. 10, 2021)

How can an infaunal brooding echinoid be maintained in the laboratory?

A case study with Cassidulus mitis (Echinoidea: Cassiduloida)

Carlos Renato Rezende Ventura

Monalisa Sousa Pinto de Oliveira

1. Museu Nacional, Universidade Federal do Rio de Janeiro. Departamento de Invertebrados, Laboratório de

Echinodermata. rventura@mn.ufrj.br (*Correspondence), monalisasousapinto2009@hotmail.com

Received 01-IX-2020. Corrected 20-X-2020. Accepted 28-XI-2020.

ABSTRACT

Introduction: Cassiduloids play a prominent role in echinoid evolutionary history because they probably are the

ancestral group of clypeasteroids. Some extant species are brooding and rare in the environment. Consequently,

there are no studies on their maintenance in the laboratory. Objective: Establish an efficient aquarium system

for C. mitis, endemic to Brazil, for ontogenetic studies. Methods: Four aquarium systems were built, with 3

replicates each one: (1) with seawater flow [F]; (2) with seawater flow and air injection into sediment [FA];

(3) without seawater flow but with air injection into the sediment [A]; and (4) without both seawater flow and

air injection into the sediment [C]. Each experimental aquarium (three per treatment) had two adults. Each of

the two sets of experiments lasted about 60 days. Results: We observed low mortality in the first 30 days in

all systems and, after 30 days, it was higher in those with air-pumped into the sediment (system A in the first

set of experiments, and system FA in the second one). Conclusions: For experiments lasting 30 days, our four

systems are suitable. For longer periods, we recommend aquaria with seawater flow and without air-pumps into

the sediment.

Key words: experimental model-species, closed aquarium system, survival rate, sand-bottom, echinoid

deposit feeder.

Rezende Ventura, C.R., & Pinto de Oliveira, M.S. (2021).

How can an infaunal brooding echinoid be maintained

in the laboratory? A case study with Cassidulus

mitis (Echinoidea: Cassiduloida). Revista de Biología

Tropical, 69(S1), 550-557. DOI 10.15517/rbt.

v69iSuppl.1.46394

In general, brooding marine invertebrates

have a rapid direct development or an abbrevi-

ate indirect development with a non-feeding

larval stage followed by a quick metamorpho-

sis. In particular, echinoderms have these pat-

terns and are recognized as useful models for

studies on embryology, life history, evolution,

and development (Evo-Devo), among other

fields of knowledge (Raff & Byrne, 2006; Hey-

land, Schuh & Rast, 2018). However, it is not

easy to find coastal marine species considered

that are suitable good experimental models.

The most significant difficulties are their avail-

ability in the field and their maintenance in the

laboratory for enough time to record valuable

changes in morphology.

The echinoid Cassidulus mitis Krau, 1954,

belongs to the order Cassiduloida Claus, 1880,

which comprises about 68 genera and 800 spe-

cies, most of them extinct (Mooi, 1990a; Kroh

& Mooi, 2020). The first records of cassidu-

loids appear in the Lower Jurassic (about 200

m.y.a). This order reached its highest diversity

in Eocene (about 55 m.y.a.) when it represented

60% of all echinoids (Kier, 1962). The decline

of cassiduloids occurred between the Late

Tertiary and Quaternary (about 30 m.y.a.).

This diversity drop was probably related to

DOI 10.15517/rbt.v69iSuppl.1.46394

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Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 550-557, March 2021 (Published Mar. 10, 2021)

global cooling and the increase of competi-

tion with other echinoids. The decline of cas-

siduloids coincided with the rise of the sand

dollars (Clyperasteroida) and heart urchins

(Spatangoida), considered as sister-groups. The

fossil records corroborate the hypothesis of

competition, causing the diversity decrease of

cassiduloids. Mooi (1990a) states that there

are 30 extant species within the order Cassidu-

loida. The morphological similarity between

the fossil and extant species is evident (Brito

& Ramires, 1974; Brito, 1981; Smith & Bengs-

ton, 1991; Squires & Demetrion, 1995), which

means that extant cassiduloids have a highly

conserved morphology. In other words, no

significant morphological novelty has occurred

along their lineage since the Upper Creta-

cean (Smith, 2001). Therefore, the extant spe-

cies show signs of “character exhaustion,” as

defined by Wagner (2000). For these reasons,

the cassiduloids fit in the concept of “living

fossil” (Schopf, 1984; Mooi, 1990b).

Cassiduloids play a prominent role in

echinoids’ evolutionary history because they

probably are the ancestral group of the sand

dollar lineage (clypeasteroids). Among all echi-

noids, the species of Cassiduloida are unique

that have the Aristotle’s lantern only during the

early young stage (post-metamorphosis). The

morphological similarity between the lanterns

of small cassiduloids and large clypeasteroids

is the primary support for this hypothesis. In

other words, the retention of ancestral young

characters in the adult descendant species

(neoteny) supports this hypothesis. Despite

its evolutionary relevance, the loss of the

Aristotle’s lantern of extant cassiduloids still

has not been well-studied (Gladfelter, 1978;

Märkel, 1978; Contins & Ventura, 2011). The

principal cause for this knowledge gap is the

difficulty of finding dense populations in the

field and the ability to maintain cassiduloids

in the laboratory for enough time to record

all significant morphological changes during

the loss of Aristotle’s lantern. The best way

to keep young post-metamorphic cassiduloids

alive is on adult females, among their spines.

Therefore, the maintenance of adults in the

laboratory is crucial.

Cassidulus mitis is an endemic infaunal

species from Brazil (Tommasi & Lima-Verde,

1970). It occurs in a relatively high abun-

dance in one locality, and it has a continu-

ous reproductive cycle, develops yolky eggs,

embryos, and lecithotrophic larvae. Cassidulus

mitis broods its offspring among the female

spines for 18 days, when the post-metamorphic

juveniles (that have the Aristotle’s lantern)

leave their parent to the coarse-sandy environ-

ment (MacCord & Ventura, 2004; Contins &

Ventura, 2011). After this period, juveniles

continue their development, assuming an ellip-

soidal shape, and lose their lantern. As adults,

they start to consume the organic matter from

the sediment.

Therefore, Cassidulus mitis is a crucial

species of evolutionary relevance. Some previ-

ous studies have already gathered important

information about its biology. Its ready avail-

ability in a coastal area gives it the potential

to be a good species model. However, it is still

necessary to establish methods to keep it in

the laboratory. This study aims to establish an

efficient aquarium system to maintain C. mitis

in laboratory conditions for enough time for

developmental studies.

MATERIALS AND METHODS

Study Area

The population of C. mitis occurs at the

beach Praia Vermelha (22º 57’ 18” S; 43º 9’ 48”

W) located at the entrance of the Guanabara

Bay (Rio de Janeiro State, Brazil). It is a short

beach with a sharp decline in the bottom caused

by waves and periodic storms. The substrate at

the Praia Vermelha consists of homogeneous

coarse sand of rounded grains of quartz (Freire,

Santos, Fontoura, Magalhães & Grohamann,

1992). The climate in this region is typically

tropical, warm, and rainy, with two distinct

seasons, dry in autumn and winter, and rainy in

spring and summer.

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Collection of specimens, seawater,

and sediment

Specimens of C. mitis were collected from

the infralittoral zone at depths between 2 and 4

meters by apnea free-diving in May and August

2019. The temperature of the seawater was

about 22

C. The salinity was about 35 ppm. We

collected seawater and sediment just after the

collection of the sea urchins.

Aquarium systems, monitoring of survival,

seawater temperature, and salinity

Adult sea urchins were kept in four aquar-

ium systems, simulating somewhat of natural

conditions. These were four sets of experimen-

tal aquaria (plastic box of 33.5cm x 24,0cm

x 16,0cm) with seawater and a layer of 5 cm

of sediment from the Praia Vermelha. Each

system had one treatment with three replicates:

(1) Aquaria with seawater flow – [F]; (2)

Aquaria with seawater flow and air-pumped

into the sediment bottom – [FA]; (3) Aquaria

without seawater flow and with air-pumped

into the sediment – [A]; and (4) Aquaria with-

out both seawater flow and air-pumped into

the sediment – [C] (Fig. 1 and Fig. 2). The air

into sediment was provided by an air-pump

connected to porous-stone sticks (12 cm long)

buried in each aquarium’s sediment layer.

Each experimental aquarium had two adults

(a total of 12 females and 12 males, ranging

from 2.2cm to 4.6cm and 2.2cm to 3.9cm

of the longest test diameter, respectively).

Sexual differences in C. mitis can be distin-

guished by their dimorphic genital papillae in

the apical region of the test (Tripneustes-type

papillae) (Tahara, Okada & Kobayashi, 1958;

Fig. 1. Experimental design of the four aquarium systems.

(FA) Aquaria with seawater flow and air-pumped in

sediment bottom; (F) Aquaria with seawater flow; (A)

Aquaria without seawater flow and with air-pumped in the

sediment; and (C) Aquaria without both seawater flow and

air-pumped in sediment.

Fig. 2. Arrangement of the four experimental aquarium systems. A - Aquaria with seawater flow and air-pumped into the

sediment (FA); Aquaria with seawater flow (F); Aquaria without seawater flow and with air-pumped into the sediment (A);

Aquaria without both seawater flow and air-pumped into the sediment (C); Tanks (30L) (R). Numbers (1, 2, 3) represent

replicates in each treatment. B – A single flow-aquarium system. Immersed pump (P); Filters (F). Arrows represent the flow

directions. Aquaria systems developed by Renato R. Ventura.

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Pearse & Cameron, 1991). These experimental

aquaria systems are original and were devel-

oped by Carlos Renato R. Ventura.

The seawater was filtered in all treatments.

Filters of the two flowing systems (F and FA)

cleaned seawater in tanks (30L). Seawater

came to tanks from aquaria by gravitation force

and returned to each aquarium by immersed

pumps (Fig. 2). These filters had three layers

that provide mechanical (with synthetic poly-

amide fibers), chemical (with charcoal), and

biochemical filtration (with ceramic rings).

Seawater flow was supplied by immersed

pumps (with the flow rate of 400 to 1 000L/h)

connected to each tank’s filters. The average

seawater flow speed in experimental aquaria

was 5.11 cm/s (± 1.19, 1SD). In two other

systems without seawater flow (A and C), fil-

ters were inside each aquarium. Because each

aquarium had a smaller seawater volume (4L)

in these cases, a single biochemical filter at the

bottom was sufficient to keep the water clean.

Each aquarium was monitored daily dur-

ing the first week and twice a week along with

the experiments, checking sea urchin survival,

and measuring seawater temperature and salin-

ity. Two sets of experiments were performed.

Each of them lasted about 60 days.

The mean seawater temperature was 23.8

± 1.9 (mean ± 1 standard deviation). Salinity

was corrected when it was over 35 ppm.

The staff of the Unit of Environmental

Analysis (UFRJ) analyzed the substrate using

the granulometric laser diffraction method.

They also measured the percentage of organ-

ic matter in the substrate by the calcination

method. We took one sediment sample before

distributing it in each aquarium, at the begin-

ning of each set of the experiment (initial), and

other sediment samples from each aquarium

of all treatments at the end of experiments

(final samples).

Statistical Analyses

We used the Kaplan-Meier Method, the

Log-Rank test (Krebs, 2017), and the ANOVA

Linear Mixed Model to compare statistical

differences of the survival rates of C. mitis in

the four aquarium systems for the two sets of

experiments (May and August 2019). We used

the R Program (The R Project for Statistical

Computing, <www.r-project.org>) to perform

all analyses.

RESULTS

The two sets of experiments lasted about

60 days. Survival rates in the four experimental

aquaria systems were higher than 50% after the

first 30 days in both sets of experiments, except

for the FA system of the second group (Fig.

3 and Fig. 4). Most of the variation occurred

after this time (30

day). It was statistically

significant in those systems with air-pumped

into the sediment (system A in the first set

of experiments, and system FA in the second

one) (Fig. 3 and Fig. 4). The death of sea

urchins occurred independently of their size.

Two brooding females (3.4 cm and 4.9 cm in

longest diameter) were tested in the second set

of experiments.

The ANOVA Linear Mixed Model corrob-

orated the Kaplan-Meier and Log-rank analy-

ses. ANOVA results show that a significant

variation of survival rates occurred as a func-

tion of time for both sets of experiments for

all aquarium systems. Also, ANOVA identified

significant deviation from the system C (Con-

trol) of treatment A in the first group and the

treatment FA in the second set of experiments

(Supplementary Material).

Granulometric analyses classified the sedi-

ment as coarse sand and moderately sorted,

confirming the previous studies at Praia Ver-

melha. The content of organic matter in the

initial sediment sample of each set of experi-

ments was very different. It was much lower

in the first group than in the second one

(Fig. 5). The highest organic matter contents

in the sediment occurred in system F at the

end of the experimental period in both sets of

experiments (Fig. 5).

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Fig. 4. Cassidulus mitis. Kaplan-Meier survival analysis and Log-Rank test (P=0.85) for the second set of experiments.

Fig. 3. Cassidulus mitis. Kaplan-Meier survival analysis and Log-Rank test (P=0.31) for the first set of experiments.

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DISCUSSION

Like other infaunal echinoderms, Cas-

sidulus mitis consumes sediment, ingesting

the organic matter associated with the grains.

Although the feeding mechanism of C. mitis is

not well-described, it probably is similar to that

of Cassidulus caribaearum Lamarck, 1801.

It uses special podia (phyllopodia and acces-

sory podia) to gather and handle coarse sand

grains with no obvious selection for particles

(Gladfelter, 1978; Telford & Mooi, 1996).

Both species of Cassidulus live in reflective

beaches, burrowed in coarse sand between

the first layers (3-5 cm) and 20 cm below the

bottom surface (Gladfelter, 1978; C.R.R. Ven-

tura, personal observation). On beaches with

a predominantly reflective system (like Praia

Vermelha), bottom currents play a relevant role

in sediment dynamics. Rip currents, incidental

storms, and reflected waves can stir up surface

layers of the bottom, providing oxygen supply,

bringing and taking organic matter from the

substrate. Therefore, the ability to obtain food

(organic matter) and oxygen and to maintain

position within the substrate is essential for

well-being and survival in such an environ-

ment. Being a sand-ingesting deposit feeder, C.

mitis (and probably C. caribaearum) keeps its

gut full of sediment all time (C.R.R. Ventura,

personal observation) to obtain energy and

enough weight warranting stability against bot-

tom current effects.

The experimental design in this study

focused on simulating somewhat similar natu-

ral conditions in the aquarium systems, evaluat-

ing the combined and isolated effects of bottom

current and air-stirring substrate in the survival

of C. mitis. The duration of the experiments

and the contents of organic matter into the sedi-

ment seem to be the principal factors that influ-

ence the survival of C. mitis under laboratory

conditions. The four treatments are similarly

efficient during the first 30 days, but they grad-

ually lose efficiency in the subsequent days.

Probably, variation in organic matter contents

in the sediment over time caused mortality after

the first month. As an alternative solution, we

propose to replace the sand of all aquaria with

a recently collected substrate from the sample

site. However, we emphasize that organic mat-

ter contents in sediment can vary greatly in a

reflective beach, spatially and temporally. In

this study, the initial substrate samples of the

two sets of experiments varied significantly.

The oxygen concentration in interstitial

seawater is also a significant factor in the sur-

vival of infaunal sea urchins. Some individuals

of C. mitis emerged from the sediment showing

hypoactivity and died after a few hours. Proba-

bly, the low concentration of oxygen in intersti-

tial seawater caused death. Unfortunately, the

oxygen concentration in interstitial seawater

was not measured, and the cause of deaths can

only be suggested. Gladfelter (1978) described

the same behavior for C. caribaearum after 24

to 36 hours in the laboratory. As the aquarium’s

oxygen concentration was low (2.13 mL O

-1

) at that time, Gladfelter (1978) suggested

that the emergence of urchins occurred due to

hypoxia. He stated that the oxygen consump-

tion of C. caribaearum ranges from 10.2 to

23.3 µL O

g wet weight

-1

. h

-1

, varying with

the weight. Also, he showed experimentally

that respiration per unit of weight is higher

in smaller sea urchins. There are no data on

oxygen consumption of C. mitis, and for this

reason, the respiration rate of C. caribaearum

is the best reference. Injection of air into the

sediment presumably could increase the con-

centration of O

in the interstitial seawater.

Fig. 5. Mean content of organic matter in the sediment

of initial samples and each of four treatments. Blue bars

= First set of experiments; Red bars = Second set of

experiments. Variation bars = Standard deviation.

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However, the highest mortality rates occurred

in those aquaria with air-pumped into the sub-

strate (treatments A and FA). The bubbles of

air within the sediment might increase the oxy-

gen concentration but also might remove the

organic matter to the water column. It seems to

be most crucial to the survival of sea urchins.

In conclusion, the best aquarium systems

to maintain C. mitis alive in the laboratory for

a long time (about 60 days) are those with-

out air-pumped into the sediment and with

seawater flow (F), and without both flow and

air-pumped in the sediment (C). For short-term

experiments (30 days or less), all four systems

seem to be suitable. In both cases, monitor-

ing the organic matter in sediment is the best

way to determine when the sediment should

be replaced.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we

followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowled-

gements section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We are thankful to Dr. Gisela Mandali and

Dr. Vinicius Peruzzi (Department of Marine

Biology, UFRJ) for all facilities provided in

laboratory and sediment analyses, to Dr. Paulo

Cesar de Paiva (Department of Zoology, UFRJ)

for statistical assistance, and to Dr. Pablo E.

Meretta for revising the Spanish Abstract. We

are also very grateful to Dr. John Lawrence and

three reviewers for their critical comments and

suggestions that improved this article. CRRV

is thankful to the Fundação Carlos Chagas

Filho de Amparo à Pesquisa do Estado do Rio

de Janeiro for the research grant (Emergency

Support to the Museu Nacional, ref. 242060),

and the Conselho Nacional de Desenvolvim-

ento Científico e Tecnológico (CNPq) for the

research grant (ref. 310167/2018-3).

RESUMEN

¿Cómo se puede mantener un equinoide infantil en el

laboratorio? Un estudio de caso con Cassidulus mitis

(Echinoidea: Cassiduloida)

Introducción: Los casiduloides desempeñan un

papel destacado en la historia evolutiva de los equinoides

porque probablemente son el grupo ancestral de clipeas-

teroides. Algunas especies existentes son inquietantes y

raras en el medio ambiente. En consecuencia, no existen

estudios sobre su mantenimiento en laboratorio. Objetivo:

Establecer un sistema de acuario eficiente para C. mitis,

endémica de Brasil, para estudios ontogenéticos. Métodos:

Se construyeron cuatro sistemas de acuarios, con 3 réplicas

cada uno: (1) con flujo de agua de mar [F]; (2) con flujo de

agua de mar e inyección de aire en el sedimento [FA]; (3)

sin flujo de agua de mar pero con inyección de aire en el

sedimento [A]; y (4) sin flujo de agua de mar ni inyección

de aire en el sedimento [C]. Cada acuario experimental

(tres por tratamiento) tenía dos adultos. Cada uno de los

dos conjuntos de experimentos duró aproximadamente 60

días. Resultados: Observamos una baja mortalidad en los

primeros 30 días en todos los sistemas y, después de 30

días, fue mayor en aquellos con aire bombeado al sedimen-

to (sistema A en el primer conjunto de experimentos y sis-

tema FA en el segundo). Conclusiones: Para experimentos

de 30 días, nuestros cuatro sistemas son adecuados. Para

períodos más largos, recomendamos acuarios con flujo de

agua de mar y sin bombas de aire en el sedimento.

Palabras clave: modelo-especie experimental, sistema

de acuario cerrado, tasa de supervivencia, fondo de arena,

depósito alimentador de equinoides.

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