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Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(S1): 334-345, March 2021 (Published Mar. 30, 2021)
The influence of density on survival and larval development in the sea
urchin Arbacia dufresnii (Echinodermata: Echinoidea)
Florencia Belén Chaar
1,2,3
Jimena Pía Fernández
2,3
Lucas R. Sepúlveda
2,3
Tamara Rubilar
2,3
*
1. National University of Patagonia San Juan Bosco, Bv. Almte. Brown 3051, Puerto Madryn, 9120, Chubut, Argentina;
chaar@gmail.com
2. Laboratory of Chemistry of Marine Organisms. Instituto Patagónico del Mar. Faculty of Natural Sciences and Health
Sciences. National University of Patagonia San Juan Bosco. Puerto Madryn headquarters. Argentina;
jpfernandez@cenpat-conicet.gob.ar; lsepulveda@cenpat-conicet.gob.ar, rubilar@cenpat-conicet.gob.ar
(*Correspondence).
3. Biological Oceanography Laboratory- Centro para el Estudio de Sistemas Marinos- Centro Nacional Patagónico,
Centro Científico Tecnológico del Consejo Nacional de Investigaciones Científicas y Técnicas- Consejo Nacional
de Investigaciones Científicas y Técnicas de Argentina, Bv. Almte. Brown 2915, Puerto Madryn, 9120, Chubut,
Argentina.
Received 13-VII-2020. Corrected 03-VIII-2020. Accepted 18-IX-2020.
ABSTRACT
Introduction: Density is one of the critical factors in echinoderm larvae for aquaculture purposes. Echinoplutei
larvae are very sensitive to overcrowding. High culture density can lead to problems with bacteria or protozoa,
decreasing survival and generating abnormal morphotypes. Objective: To evaluate the effect of culture density
on survival and larval growth in the sea urchin Arbacia dufresnii. Methods: Two days after fertilization of A.
dufresnii we we kept treatments at 1, 3, 5 and 10 larvae.ml
-1
, with three replicates each. We recorded survival
and abnormal morphotypes periodically, as well as growth:somatic rod length, total length, and length of the post
oral arms,. we applied generalized linear models. Results: Survival is dependent on density, time and replicates,
and their interactions. Larval growth depended on density and time, also with interaction between the variables.
The treatment of 5 larvae.ml
-1
had the highest survival and larval condition. Conclusions: Larval culture of A.
dufresnii had the best results at 5 larvae.ml
-1
.
Key words: Echinoplutei, growth, morphometry, aquaculture, treatments.
Belén Chaar, F., Fernández, J.P., Sepúlveda, L.R., &
Rubilar, T. (2021). The influence of density on
survival and larval development in the sea urchin
Arbacia dufresnii (Echinodermata: Echinoidea).
Revista de Biología Tropical, 69(S1), 334-345. DOI
10.15517/rbt.v69iSuppl.1.46365
Echinoderm embryos, particularly sea
urchins and starfish, have been studied for
more than 170 years (Rubilar & Crespi-Abril,
2017). The culture is simple and has been used
as a classical animal model for embryological
studies (Williams & Anderson, 1975). More
recently, echinoderm embryos have been used
for ecotoxicology analysis (Rahman, Tsuchi-
ya, & Uechara, 2009) and molecular studies
(McClay, 2011; Hinman & Burke, 2018). Addi-
tionally, the development of simple protocols
facilitates the aquaculture of the entire life cycle
of sea urchins. This progress has been crucial in
the further development of aquaculture and in
DOI 10.15517/rbt.v69iSuppl.1.46365
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Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 334-345, March 2021 (Published Mar. 30, 2021)
the implementation of repopulation projects in
areas affected by overexploitation, or by mass
mortality, as found in Japan, Chile and China,
among other areas (Unuma, Sakai, Agatsuma,
& Kayaba, 2015; Brady & Scheibling, 2006).
As a result, advances in this area are of major
interest for research, conservation and aquacul-
ture purposes.
The echinoderm larvae culture is crucial to
any conservation or aquaculture project. There
is a need of having certain, optimal growing
conditions in order to obtain good results in
the successful production of post settled juve-
niles. Variables, such as temperature, salin-
ity (Astudillo, Rosas, Velásquez, Cabrera, &
Maneiro, 2005; Domínguez, Rosas, Velásquez,
Cabrera, & Mata, 2007), feeding, and larval
culture density (Astudillo et al., 2005; Buitrago
& Lodeiros-Seijo, 2005) are among the most
important factors.
There exists a wide variety of values
in the optimal culture density depending on
the species, with values ranging from 0.025
to 3 larvae.ml
-1
(Buitrago & Lodeiros-Seijo,
2005; Salas-Garza, Carpizo-Ituarte, Parés-Sier-
ra, Martínez-López, & Quintana-Rodríguez,
2005; Kalam-Azad, Mckinley, & Pearce, 2010;
Díaz-Pérez & Carpizo-Ituarte, 2011). High
culture densities can affect the quality of the
water, increasing the risk of contamination
due to the increase in organic matter, as well
as the increase in waste metabolic substances
(Lamare & Barker, 1999; Salas-Garza et al.,
2005; Domínguez et al., 2007; Clark, Lamare,
& Baker, 2009; Kalam-Azad et al., 2010). High
densities can also generate stress or competition
for space or food, affecting the normal develop-
ment of the organisms (Pechenik, 1999; García
et al., 2005) Inadequate density cultures usually
translate into decreased survival, changes in the
growth of organisms, and/or a higher incidence
of abnormal phenotypes, such as the absence
of arms or very small bodies with very long
arms (Clark et al., 2009; Kalam-Azad et al.,
2010). However, considering the natural mor-
tality during the larval stage and the specific
culture conditions, it is necessary that the initial
quantity of organisms is sufficient to be able to
obtain a production of juveniles justifying the
required investment and efforts (Stotz, 2014).
For aquaculture purposes, the maximum viable
density of a species is of particular importance.
In addition, when analyzing a culture, it is
important to consider the morphometrics of
the larvae, both in terms of size and as well as
in terms of the proportion of its structures as
these can provide information on the culture
conditions and the effects on development.
To estimate larval growth, both PO (post oral
arms) and TL (total length) of sea urchin larvae
have been two of the most frequently used
indicators in recent years. The PO is one of the
most frequently used measures as it is, gener-
ally, very sensitive to changes in environmental
variables (Fenaux, Strathmann, & Strathmann,
1994; Hart & Strathmann, 1994; Eckert, 1998;
Jimmy, Kelly, & Beaumont, 2003; Cárcamo,
Candia, & Chaparro, 2005; Domínguez et al.,
2007; Clark et al., 2009; Rosas, Velásquez,
Fernández, Mata, & Cabrera, 2009; Adams,
Sewell, Angerer, & Angerer, 2011; Fernández,
2019). At the same time, measuring the length
of the somatic rod (SO), can be an impor-
tant indicator of growth parameters related
to ambient factors (Clark et al., 2009; Feu-
nax et al., 1994).
The sea urchin Arbacia dufresnii is abun-
dant along the Argentinian and Chilean coasts,
inhabiting coastal areas from Río de la Plata
(35° S) in the Southwest Atlantic to Puerto
Montt (42° S) in the South Pacific, including
the Malvinas Islands (Bernasconi, 1966). This
species has been the subject of several scientific
studies. Specific ecological aspects have been
described, such as their abundance, distribu-
tion, role in the ecosystem and diet in different
environments (Vásquez, Castilla, & Santelices,
1984; Larraín, Mutschke, Riveros, & Solar,
1999; Penchaszadeh & Lawrence, 1999; Zaixso
& Lizarralde, 2000; Teso, Bigatti, Casas, Piriz,
& Penchaszadeh, 2009; Newcombe, Cárdenas,
& Geange, 2012; Brogger et al., 2013; Castro,
2014; Epherra, 2016; Epherra, Martelli, Mor-
san, & Rubilar, 2017). Descriptions of certain
physiological aspects of adult individuals have
been described, such as the reproductive cycle
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and gonadal composition (Brogger & Iva-
nov, 2010; Epherra et al., 2015a; Epherra et
al.,2015b; Parra et al., 2015; Zárate, Díaz de
Vivar, Ávaro, Epherra, & Sewell, 2016). Addi-
tionally, specific aspects of the early stages of
the life cycle of this species have also been
described, such as fertilization under controlled
conditions (Fernández, Epherra, Sepúlveda, &
Rubilar, 2019), complete embryonic develop-
ment and specific stages of larval development
(Bernasconi, 1942; Brogger, 2005; Fernández,
2019). Furthermore, there have been descrip-
tions of the response of the larvae to ocean
acidification (Catarino, De Ridder, Gonzalez,
Gallardo, & Dubois, 2012) and the monitoring
of development in natural conditions (Kino,
2010). These studies have served as a basis
for the exploration of the aquaculture potential
of the species (Rubilar et al., 2016). However,
there are, still, few studies which have succeed-
ed in determining the optimal larval culture
density under laboratory conditions. Neither
are there any large number of studies defining
and clarifying the possible consequences of
culturing in a variety of densities. The aim of
this study is to evaluate the influence of culture
density on survival and on the larval develop-
ment of the sea urchin A. dufresnii which is a
new species within aquaculture.
MATERIALS AND METHODS
Spawning and fecundity: The adult spec-
imens of A. dufresnii, belonging to the brood-
stock of the experimental aquarium of the
Centro Nacional Patagónico, Consejo Nacional
de Investigaciones Científicas y Técnicas de
Argentina (CONICET), were the subject of
investigation. For the induction of spawning,
four females and three males of A. dufresnii
were injected with 0.3 ml of 0.5 M KCl solu-
tion (Strathmann, 1987). The female gametes
were collected individually on an aqueous
medium and, were, then, mixed in a single
container and quantified in triplicate. Male
gametes were dry harvested in a single con-
tainer and kept on ice until used to preserve
their viability (Ettensohn, Wessel, & Wray,
2004; Strathmann, 2014; Ettensohn, 2017). A
total of 800 000 eggs were placed in a liter of
sea water filtered up to 1 µm and sterilized with
UV light. Fertilization was carried out by using
a 1:100 000 v/v dilution of sperm according
to Fernández et al. (2019). After 30 min, the
percentage of fertilization success was verified
and calculated under a light microscope (Leica
DM 2500) on the basis of observing the fertil-
ization membrane in the eggs.
Embryo culture: The fertilized eggs were
kept for 48 h with constant and gentle aeration,
at a temperature of approximately 17 °C and at
a salinity of 34-35 ppm. At 24 h after fertiliza-
tion, the culture volume was tripled in order to
decrease the density of developing organisms.
After 72 h, the larvae in the prism stage were
transferred to a new container, quantified, and
the corresponding dilutions were undertaken
for each treatment.
Larvae culture: Four treatments with
varying densities of larval culture, were pro-
duced. The treatments involved included:1
larva.ml
-1
, 3 larvae.ml
-1
, 5 larvae.ml
-1
, and 10
larvae.ml
-1
. All of the treatments were executed
in triplicate and the larvae were kept in sealed
systems consisting of 1 liter glass containers.
The larvae were cultured for 14 days, which
corresponds to 2 to 14 days postfertilization
(DPF) of development. This took place under
controlled conditions and similar water qual-
ity. The larvae were fed, daily, with a mix of
microalgae Tetraselmis suecica and Isochry-
sis galbana, at an initial rate of 10 000, and
this was gradually increased every two days
to reach 40 000 cells.larvae
-1
(Unuma et al.,
2015). Sea water changes took place every
48 h, removing 50 % of the total volume of
each container, using filters with a 50 µm
mesh to avoid the loss of larvae, and adding
acclimatized sea water (water at the same tem-
perature as that of the crops to be replaced).
With each water exchange, pH, temperature,
salinity and nitrogen compounds were mea-
sured in each specific replica. The final water
exchange was based on applying commercial
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tests for nitrates (Nutrafin), nitrites (Nutrafin)
and ammonia (Tetra).
Survival: Five counts were undertaken
during the experiment (that is, every 3 days).
Using a zoom microscope (Stemi 2000, Carl
Zeiss) and using a bogorov chamber, the num-
ber of larvae observed in a 30 ml aliquot for
each treatment and replicates was quantified.
Only organisms with normal morphology and
vitality were taken into account on the basis of
the descriptions of Fernández et al. (2019). The
ability to swim, the emergence of typical struc-
tures during larval development, homogeneity
of the development of each treatment and the
left-right symmetry in individuals, was clearly
observed. The survival percentage was calcu-
lated at: Survival = (number of larvae at a given
point in time t + 1) / (initial larval number at a
given point in time t) * 100.
Growth: In order to study larval growth
over time, photographs of between three to five
larvae were taken of each replica, every three
days. For this purpose, a Leica DM 2500 bright
field microscope and imaging software for
microscopy were used. Then, using the ImageJ
software, the length of the somatic rod (SO)
(length from the apical end to the beginning of
the post oral arm), the length of the post oral
arm (PO) (length from the end of the somatic
rod to the end of the post oral arm), and the
total length (TL) (length from the apical end of
the larva to the end of the post oral arm) were
measured (Fig. 1). Only larvae with normal
morphology and vitality were considered on
the basis of the descriptions of Fernández et
al. (2019).
Data analysis: As a first step, a graphical
exploration, based on the scatter and residual
plots of the data, was performed in order to
understand the relationship between the surviv-
al, response and the growth the variables (TL,
PO and SO), as well as to explain the explana-
tory variables (time -as DPF-, and density).
To analyze the effect of density on survival, a
Generalized Linear Model (GLM) was applied.
Different replicates were included in the model
analysis in order to evaluate method error and
in order to analyze whether the replicates pre-
sented similar values. Twelve different models,
starting with a null model (simpler model with-
out any explanatory variable) were proposed,
and the complexity of the models resulted in
a complex triple interaction model, between
the explanatory variables referred to as DPF,
replicates and density. To analyze growth over
time after fertilization (DPF), a GLM was also
applied. We proposed five models starting with
a null model, whereby we increased complex-
ity to result in a double interaction model tak-
ing place between the explanatory variables,
DPF, and density. The best model was selected
on the basis of the Akaike criterion, taking into
account the residual analyzes, the explained
variance (deviation) and the principle of par-
simony or normality (Hurvich, Simonoff, &
Tsai, 1998). The Kruskal-Wallis non-paramet-
ric analysis of variance tests was used to deter-
mine the significance between densities. This
was accompanied by peer reviews in order to
identify the association between treatments.
Fig. 1. Morphometric measurements to evaluate larval
growth. SO = somatic rod. PO = post oral arm rod. TL =
total larval length. Scale bar = 100 µm.
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All GLM analyzes were performed with the
free software RStudio (version 3.5.1), and for
the Kruskal-Wallis tests the software InfoStat
was used. For all of the analyses, the level of
significance applied was P value < 0.05.
RESULTS
Survival: Larval survival (%) decreased
over time differently for each density treat-
ment. The lowest values were for the lowest
density (1 larva.ml
-1
), decreasing to 70 % in the
first sampled period (5 DPF), and decreasing
to less than 20 % by the end of the experiment
(14 DPF). The intermediate density treatments
recorded a survival percentage between 70 %
and 85% up to 11 DPF, and a notable decrease
by 14 DPF (3 larvae.ml
-1
). A high, and more
stable, survival percentage of approximately
82% throughout the entire sample period was
found in the 5 larvae.ml
-1
treatment, which
gradually decreased to be around 54 % by 14
DPF. The higher density treatment (10 larvae.
ml
-1
) showed high survival, exceeding 90 %,
until 11 DPF, when it started to decrease to
approximately 40 % by 14 DPF (Fig. 2).
Table 1 shows the results of the GLM
analysis for survival. The most complex model,
which included the triple interaction between
density, time (DPF) and replicates, appeared to
be best matched with the data.
Abnormal larval development: During
the experiment, morphologically normal lar-
vae, and larvae with varying abnormal morpho-
types were found (Fig. 3). The most common
morphotype is represented in Fig. 3A. On the
other hand, morphologically abnormal larvae
can be larvae with very small post oral arms
(Fig. 3B), larvae with a wide angle between
post oral arms (Fig. 3C), larvae with crossed
post oral arms (Fig. 3D), and larvae with
square apical end (Fig. 3E). These abnormal
morphotypes appeared on 8 DPF, registering
Fig. 2. Arbacia dufresnii larval survival percentage through time as regards four different
densities: 1, 3, 5 and 10 larvae.ml
-1
(mean ± standard error).
TABLE 1
Generalized Linear Model analysis for larval survival
of Arbacia dufresnii. In bold, the model with the best fit
according to the Akaike information criterion (AIC).
DPF = days post fertilization.
Model for survival d.f. Akaike
1. Null model 1 24 7544.75
2. Density 4 88 215.902
3. DPF 7 17 4118.85
4. Replicates 2 10 2241.47
5. Density + DPF 10 14 790.003
6. Density + replicates 5 88 212.401
7. DPF + replicates 8 28 815.572
8. Density * DPF 28 7 333.772
9. Density * replicates 8 87 362.07
10. DPF * replicates 14 26 581.575
11. DPF + replicates + Density 11 14 025.098
12. DPF * Replicates * Density 56 3 558.037
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approximately 10 % in all treatments. Howev-
er, the percentage of abnormal larvae observed
throughout the experiment varied with density,
reaching more than 35 % in the lowest density
(1 larva.ml
-1
) by day 14, less than 20 % in inter-
mediate densities (3 larvae.ml
-1
) and less than
10 % in the highest density (5 and 10 larvae.
ml
-1
) (Fig. 4).
Growth: Larvae size was similar in all
densities at the initiation of the experiment.
With time (DPF), the PO and TL increased in
all densities but showed differential growth,
both in the length of PO (Fig. 5A) and in TL
(Fig. 5B). The differences in larvae growth
were more evident by the end of the experi-
ment, when both PO and TL, showing the low-
est density (1 larva.ml
-1
), were two-fold higher
than in the higher density (5 and 10 larvae.
ml
-1
), evidencing significant differences (KW =
22.49, P = 0.0001, and KW
= 28.25, P < 0.0001,
respectively). There was no change in SO over
Fig. 3. Larval morphotypes of Arbacia dufresnii taken with Leica DM 2500 software (10X magnification).
A. Normal. B-E. The most representative abnormal larval morphotypes. Scale bar = 100 µm.
Fig. 4. Larvae with abnormal morphotypes (%) during the experiment (mean ± standard error).
On days 2 and 5 after fertilization, larvae did not present abnormal morphotypes.