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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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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.

-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.

-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.

-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.

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time; however, there were significant differenc-

es between densities at 8 and 14 DPF (KW

8.66, P = 0.0334, and KW = 14.28, P = 0.0019,

respectively) (Fig. 5C). Throughout the sample

period, there were slight variations in the mor-

phometric index as regards the different larvae

densities, and these differences become more

evident by 14 DPF (Fig. 5D). These differences

indicate that there is a relationship between the

treatments studied and the degree of growth of

the post oral arms.

Table 2 shows the results of the GLM

analysis as regards growth. The most complex

model, which includes the interaction between

density and time (DPF), was the best fit for the

data concerned.

DISCUSSION

The optimal culture density found for A.

dufresnii larvae was higher than that usually

Fig. 5. Arbacia dufresnii larvae growth. A. Post oral arm rod larval length (PO). B. Total larval length (TL). C. Somatic rod

larval length (SO). D. Morphometry index. (Mean between 3 and 5 measures ± Standard error) (***and * denotes significant

differences P ≤ 0.001 and P = 0.0334, respectively).

observed for other species. In Evechinus chlo-

roticus, Lytechinus variegatus, Paracentrotus

lividus and Loxechinus albus it was suggested,

to increase survival, to culture with densities

of 1 larva.ml

-1

, 0.25 or 0.5 larvae.ml

-1

, 0.06

larvae.ml

-1

and 0.7 larvae.ml

-1

respectively

(Buitrago & Lodeiros-Seijo, 2005; Salas-Garza

et al., 2005). However, for Arbacia stellata a

similar density was proposed (4 larvae.ml

-1

)

(Díaz-Martínez, 2019). Differences in larvae

size among species may be related to the den-

sity found in cultures. Evechinus chloroticus

two-armed pluteus larvae exceeds the 200

µm, L. variegatus, two-armed pluteus larvae

is around 400 µm, P. lividus four-armed plu-

teus larvae that exceeds 600 µm, and L. Albus

reaches approximately 500 µm (Fenaux et

al., 1994; Lamare & Baker, 1999; Buitrago &

Lodeiros-Seijo, 2005; Cárcamo et al., 2005).

Arbacia dufresnii at the same stages of devel-

opment, presents larvae of similar size than

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A. stellata (Díaz-Martínez, 2019). Size is an

important factor in larvae culture, since at the

same density the area occupied by each larva

will be different according to the larvae´s size.

In this way, at high culture density, collisions

between larvae can occur, increasing stress

and affecting their development (Buitrago &

Lodeiros-Seijo, 2005). Therefore, it is prob-

able that A. dufresnii can be culture at higher

densities without affecting survival due to its

small size.

Abnormal morphotypes decreased survival

in A. dufresnii since these morphotypes do not

prosper. At 8 DPF, 10 % of abnormal morpho-

types were found in all densities. However, the

extreme values of density (1 larva.ml

-1

and 10

larvae.ml

-1

) showed at the end of the experi-

ence, the highest frequency in abnormal larval

morphotypes and the lowest survival. The

presence of these morphotypes in A. dufresnii

larval culture was first described by Bernasconi

(1942) and later on by Brogger (2005), who

observed changes in morphology due to stress

by increased temperature. In this way, the

abnormal morphotypes may be related to an

increase in the stress level in the extreme den-

sity cultures. The GLM analysis indicated that

survival is affected by the interaction of three

factors: time (DPF), density, and the replica-

tions within each treatment. The fact that the

replicas were selected to be included in the

model analysis indicates that the replicas are

of utmost importance. Each culture replica sig-

nificantly impacts the overall results in terms

of larval survival, underlying the importance

of the microenvironmental conditions (slight

changes in temperature, light exposure, oxygen

availability, etc.) in each culture and not only

density as the main factor.

Morphometric measurements can reflect

the most striking changes in organisms over

time. In this study we focus on the space occu-

pied by each larva in the culture and its effect

and differential growth was observed between

densities. Again, the extreme densities showed

the greatest differences. In the lowest-density

treatment both the PO and the TL of the larvae

were higher than the rest of the treatments. On

the contrary, in the highest-density treatment,

both the PO and the TL were lower than the rest

of the treatments. No differences were found in

SO due to density, however SO is affected by

variations in other variables such as pH in other

species (Clark et al., 2009; García, Clemente,

& Hernández, 2018). The morphometric index

is useful to analyze the percentage of larvae

that occupy the post oral arms. As a result, this

can provide information on the shortening or

elongation of these arms at different densities

and regardless of size. In this study, at low

densities, the post oral arms are the longest

structure in the larvae. However, at intermedi-

ate densities, the length of the post oral arms is

not greater than the length of the SO, indicating

a more proportional larva. The GLM analysis

in all larval measurements showed that the best

model was the one that considered the effect of

the interaction between time (DPF) and density

(treatments). The time component was expect-

ed, as the larvae develop with it and, in the

process, grow and change their morphometry.

TABLE 2

Generalized Linear Model analysis for larval growth of

Arbacia dufresnii. In bold, the model with the best fit

according to the Akaike information criterion (AIC). DPF

= days post fertilization. PO = post oral larval length. TL

= total larval length. SO = somatic rod larval length.

Model for PO d.f. Akaike

1. Null model 1 8 218.877

2. Density 4 7 973.662

3. DPF 4 6 594.189

4. Density + DPF 7 6 152.789

5. Density * DPF 16

5 606.735

Model for TL d.f. Akaike

1. Null model 2 3 882.068

2. Density 5 3 874.307

3. DPF 5 3 782.868

4. Density + DPF 8 3 756.017

5. Density * DPF

17 3 696.84

Model for SO d.f. Akaike

1. Null model 1 2 435.063

2. Density 4 2 433.763

3. DPF 4 2 437.452

4. Density + DPF 7 2 435.777

5. Density * DPF 16 2 439.823

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Together, these results indicate that the higher

the density of the culture, the smaller the size

of the larvae.

It is important to note that food rations

are also crucial, since inadequate food rations

translate into development arrest (Astudillo

et al., 2005), as well as changes in the size

of the post oral arms (Hart & Strathmann,

1994). Adequate density is crucial to avoid

food competition and allow normal develop-

ment to occur (Kalam-Azad et al., 2010), since

overcrowding can drive competition (García

et al., 2005; Kalam-Azad et al., 2010). How-

ever, in this study we provided food per larva,

and not per unit of culture volume, in order to

avoid food competition. This feeding regime

eliminates food competition, but it modifies

the availability of food as it changes the den-

sity of microalgae per unit culture volume

(Unuma et al., 2015). This differentiation in

the availability of food may produce a greater

expenditure of capture effort, particularly in

low density treatments. When availability of

food is high, the post oral arms are short and

energy is invested in accelerating development

(Byrne et al., 2009). In contrast, when food

is scarce, the post oral arms tend to lengthen

to improve swimming and increase food cap-

ture (Fenaux et al., 1994; Hart & Strathmann,

1994; Byrne, Sewell, & Prowse, 2008; Adams

et al., 2011). However, food availability is

not the only cause of this effect. A nutrition-

ally insufficient diet can also have the effect

of lengthening the post oral arms (Cárcamo et

al., 2005). We have shown that density is an

important factor in A. dufresnii larvae culture,

and seems to be species specific. We also found

that the feeding regime used in this study may

have influenced in a greater manner the larval

growth and survival. This factor was especially

visible at extreme densities, demonstrating the

profound effects of food availability in larvae

culture. Therefore, additional experiments will

be necessary in order to differentiate the effect

of culture density and the effect of food avail-

ability on larval development.

Combination of variables appear to explain

survival and growth in the different treatments

in A. dufresnii. On one hand, the lowest density

treatment probably produces low food avail-

ability per unit of volume, evidenced by the

larger size of the post oral arms developed by

the larvae, and a high morphometric index.

In addition, this stressor may explain the low

survival rates found since the beginning in

this treatment, and the increase in the occur-

rence of abnormal morphotypes along days.

On the other hand, the higher density treatment

probably produces overcrowding but not low

food availability. This stressor may explain the

smaller larvae size, and a low morphometric

index, followed by a drop-in survival after

11 DPF. This decrease in survival is probably

related to poor water quality, increased organic

matter and metabolic waste substances due

to the high density and food availability, as

reported by other authors (Lamare & Barker,

1999; Salas-Garza et al., 2005; Domínguez et

al., 2007; Clark et al., 2009; Kalam-Azad et al.,

2010). Culture water exchange was made every

48 h. And even though culture water quality

was evaluated with non-analytical methods,

we cannot rule out that water quality affected

the experiment, since larvae have a higher

sensitivity (Lamare & Barker, 1999; Salas-

Garza et al., 2005; Domínguez et al., 2007;

Clark et al., 2009; Kalam-Azad et al., 2010).

Since A. dufresnii has a relatively small larva,

crowding only began to be notable at a high

concentration (10 larvae.ml

-1

). To this species,

intermediate density (5 larvae.ml

-1

) has shown

to be optimal to balance the availability of

food and overcrowding. This treatment also

presents an intermediate larval size and a low

level of abnormal morphotypes in the culture,

increasing survival chances. However, since

this experiment was not done until larvae were

ready to settle, optimal density might change in

more developed larvae.

This research represents the first study

to analyze the larval culture density of A.

dufresnii and its relevant information since

this species has become the focus of a new

aquaculture venture.

343

Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 334-345, March 2021 (Published Mar. 30, 2021)

ACKNOWLEDGMENTS

We are grateful to the diver Ricardo Bebo

Vera for the collection of the sea urchins, to the

Technician Mariano Moris for helping with the

experiments and to Kathleen C. Anderson for

the revision of English language. This work

was undertaken with funds from PIP 0352/14,

PICT 2018-1729. The sea urchins were col-

lected by the Provincial Permit N°586/18.

RESUMEN

Influencia de la densidad en la supervivencia y el

desarrollo larvario del erizo de mar Arbacia dufresnii

(Echinodermata: Echinoidea)

Introducción: La densidad es uno de los factores

críticos para las larvas de equinodermo para fines de acui-

cultura. Las larvas de Echinoplutei son muy sensibles al

hacinamiento. Una alta densidad de cultivo puede provocar

problemas con bacterias o protozoos, disminuyendo la

supervivencia y generando morfotipos anormales. Obje-

tivo: Evaluar el efecto de la densidad de cultivo sobre la

supervivencia y el crecimiento larvario del erizo de mar

Arbacia dufresnii. Métodos: Dos días después de la fer-

tilización de A. dufresnii se mantuvieron los tratamientos

a 1, 3, 5 y 10 larvas.ml

-1

, con tres repeticiones cada uno.

Registramos la supervivencia y los morfotipos anormales

periódicamente, así como el crecimiento: longitud de

la varilla somática, longitud total y longitud de los bra-

zos posorales. aplicamos modelos lineales generalizados.

Resultados: La supervivencia depende de la densidad, el

tiempo y las réplicas y sus interacciones. El crecimiento

larvario dependió de la densidad y el tiempo, también de la

interacción entre las variables. El tratamiento de 5 larvas.

-1

tuvo la mayor supervivencia y condición larvaria.

Conclusiones: El cultivo larvario de A. dufresnii tuvo los

mejores resultados con 5 larvas.ml

-1

Palabras clave: Echinoplutei, crecimiento, morfometría,

acuicultura, tratamientos.

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