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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)
Does the righting behaviour effectively reflect stress
in Arbacia dufresnii Arbacioida: Arbaciidae)?
Florencia Chaar1, 2; https://orcid.org/0000-0002-0841-2945
Tamara Rubilar1, 2; https://orcid.org/0000-0003-1728-3273
Augusto C. Crespi-Abril1, 2*; https://orcid.org/0000-0002-6278-2787
1. National University of Patagonia San Juan Bosco, Laboratory of Chemistry of Marine Organisms, Instituto Patagónico
del Mar, Faculty of Natural Sciences and Health Sciences, Boulevard Almirante Brown 3051, Puerto Madryn,
9120, Chubut, Argentina; fchaar@cenpat-conicet.gob.ar, rubilar@cenpat-conicet.gob.ar, crespi@cenpat-conicet.gob.ar
(*Correspondence)
2. 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, Boulevard Almirante Brown 2915, Puerto Madryn, 9120, Chubut, Argentina.
Received 13-VI-2023. Corrected 18-XII-2023. Accepted 07-I-2024.
ABSTRACT
Introduction: Righting behaviour has been used as a health indicator in response to stressor variables. Using this
parameter in aquaculture could help to reduce mortality and improve welfare in the sea urchin Arbacia dufresnii
culture.
Objective: The purpose of this study was to determine the effect of sex, diameter, and three stressor factors on
the righting behaviour of the sea urchin A. dufresnii.
Methods: A total of 300 animals were evaluated for complete righting behaviour (CRB) time, with 100 of them
also recording half righting behaviour (HRB) time. Three stressors were applied to the animals: serial repetitions
(three successive turnings), temperature (24-hour shock), and spawning induction with KCl injection. A stop-
watch was used to record the time, and a precision calliper was used to measure the diameter.
Results: Righting time was discovered to be diameter dependent but sex independent. The upper temperature
limit of 19 °C had a significant effect on righting behaviour compared to 16 °C and 13 °C with CRB times up to
150 seconds. Serial repetitions and spawning had no significant effect. However, based on the recorded times, it
can be deduced that spawning had an effect on the health of the animals, with CRB times of up to 150 seconds
compared to the control, with lower times.
Conclusions: Complete righting behaviour appears to be an optimal indicator for evaluating the health and
condition of the sea urchin A. dufresnii, but more tests would be performed to confirm the effect of the control
treatment on post-spawning stress.
Key words: righting time, righting behaviour, stressors, sea urchin, Arbacia.
RESUMEN
¿El comportamiento de enderezamiento refleja efectivamente el estrés
en Arbacia dufresnii (Arbacioida: Arbaciidae)?
Introducción: El comportamiento de enderezamiento se ha utilizado como indicador de salud en respuesta a
variables estresantes. La aplicación de este parámetro en acuicultura podría ser beneficiosa para reducir la mor-
talidad y mejorar el bienestar en el cultivo del erizo de mar Arbacia dufresnii.
https://doi.org/10.15517/rev.biol.trop..v72iS1.59015
SUPPLEMENT
2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)
INTRODUCTION
Since the 18th century, sea urchins have
been used as models in scientific studies
(Crespi-Abril & Rubilar, 2018; Dufossé, 1847).
Recently, there has been a growing recognition
of the importance of including all inverte-
brates, including echinoderms, under specific
ethical regulations (Crespi-Abril & Rubilar,
2021). Welfare is a broad concept that provides
information about the state and conditions of
animals in a variety of situations (Broom, 1991;
Damián & Ungerfeld, 2013). It can be measured
using specific indicators, including behaviour.
Indicators are conceptual tools for quan-
tifying dimensions and producing numerical
results that can be applied to any animal phy-
lum (Damián & Ungerfeld, 2013). This tool
must meet certain criteria, including repre-
senting correlations between variables, being
geographically and temporally contextualised,
being simple to collect and interpret, and being
based on valid scientific research (Tilbury,
2007). In the context of aquaculture, where
animal welfare and health are critical to sur-
vival, developing control measures and dis-
ease prevention is critical. High crop density,
poor water quality, incorrect or insufficient
nutrition, incorrect temperature, and the pres-
ence of pathogens, among other factors, have
been a significant source of illness and mass
mortality in aquaculture, resulting in signifi-
cant losses (Food and Agriculture Organization
[FAO], 2016).
In echinoderms, the mechanism of right-
ing behaviour involves a 180° turn on the axis
to return to the original position, with the oral
face facing the substrate and the aboral face
facing the medium (Challener & McClintock,
2017) (Fig. 1). This is a simple nervous reflex
action studied in sea urchins and sea stars to
better understand their behaviour (Lawrence &
Cowell, 1996). It appears to be essential for pro-
tection against predators and strong surges, and
it is important for sea urchin fitness (Brothers &
McClintock, 2015; Percy, 1973; Shi et al., 2018).
Because sea urchins lack muscular tissue,
they rely on their tube feet to propel them for-
ward (Binyon, 1972). As a result, their ability to
right themselves is determined by their physi-
cal condition and neuromuscular coordination
(Himmelman et al., 1984). Furthermore, as
mentioned by Lawrence (1975) and Challener
& McClintock (2017), sea urchins with larger
spines have an advantage in recovering their
position, with shorter righting times compared
to those with smaller spines. The speed of a sea
urchins righting action can be used to predict its
physiological activity, health, and overall state
Objetivo: El objetivo de este estudio fue evaluar el efecto del sexo, el diámetro y tres factores estresantes sobre el
comportamiento de enderezamiento del erizo de mar A. dufresnii.
Métodos: Se midieron un total de 300 animales para evaluar el tiempo de comportamiento de enderezamiento
completo (CRB) y 100 de ellos también registraron el tiempo de comportamiento de enderezamiento medio
(HRB). Se aplicaron tres factores estresantes a los animales: repeticiones seriadas (tres giros sucesivos), tempera-
tura (shock de 24 horas) e inducción del desove con inyección de KCl. El tiempo se midió con un cronómetro y
el diámetro con un calibre de precisión.
Resultados: El tiempo de enderezamiento resultó ser dependiente del diámetro, pero independiente del sexo. El
límite superior de temperatura de 19 °C tuvo un efecto significativo en el comportamiento de enderezamiento en
comparación con las temperaturas de 16 °C y 13 °C, con tiempos de CRB de hasta 150 segundos. Las repeticiones
seriadas y el desove no tuvieron un efecto significativo. Sin embargo, con base en los tiempos registrados, se puede
deducir que el desove tuvo un impacto en la salud de los animales con tiempos de CRB de hasta 150 segundos en
comparación con el control, con tiempos inferiores.
Conclusiones: El comportamiento de enderezamiento completo (CRB) parece ser un indicador óptimo para
evaluar la salud y condición del erizo de mar A. dufresnii, sin embargo sería óptimo realizar más ensayos para
corroborar el efecto del tratamiento control con respecto al desove.
Palabras clave: tiempo de enderezamiento, comportamiento de enderezamiento, estresores, sea, erizo de mar,
Arbacia.
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(Himmelman et al., 1984; Percy, 1973). Various
factors, including diameter, salinity, acidifica-
tion, light intensity, covering material, tempera-
ture, and others, can influence this behaviour
(Brothers & McClintock, 2015; Dihel et al.,
1979; Hamilton, 1922; Kleitman, 1941; Sun et
al., 2019; Taylor et al., 2014). Furthermore, the
most commonly used method for spawning
sea urchins involves an invasive puncture in
the peristomal membrane to inject KCl 0.5 M,
but the potential stress, mortality, and effect on
righting behaviour caused by this method have
not been studied (Strathmann, 1987).
Furthermore, there are two methods for
measuring righting behaviour in sea urchins:
half righting behaviour (HRB), which involves
reaching 90° of the surface, and complete right-
ing behaviour (CRB), which involves the ani-
mals returning to their natural state, with their
oral side on the substrate (Fig. 1). For a long
time, these two techniques have been described
and used with a variety of species, including
Lytechinus variegatus (Lamarck, 1816), Stron-
gylocentrotus purpuratus (Stimpson, 1857), and
Strongylocentrotus droebachiensis (O.F. Müller,
1776) (Challener & McClintock, 2017; Son-
nenholzner et al., 2010). The reasons for using
one parameter over another in various studies
are rarely clarified, nor is an analysis of the
benefits and drawbacks of using one parameter
over another.
There have been no previous studies of
the righting behaviour of the sea urchin spe-
cies Arbacia dufresnii (Blainville, 1825) in
nature or in the laboratory. The study of this
species’ righting behaviour in response to
stressors can provide insights into the health
and welfare of sea urchins that can be used in
aquaculture systems.
MATERIALS AND METHODS
Animal collection and acclimatization: A
total of 300 individuals of A. dufresnii were col-
lected by scuba diving from the Golfo Nuevo,
Chubut, Argentina (42.70° S & 65.60° W) dur-
ing the winter of 2022. They were transferred to
the experimental aquarium of the technology-
based company EriSea S.A in 10 buckets of
20L with 30 animals each one, without added
oxygen, in seawater of the collection site. Trans-
portation lasted approximately 15 minutes by
car from the collection site to the experimental
aquarium. The sea urchins were acclimated for
seven days at 9 ± 1 °C (the same temperature at
which they were collected) to ensure that their
digestive systems were completely empty and
that they were all in the same conditions. They
were kept in pond tanks of 570 L with filtered
sea water at a density of 2 animals per litre, with
oxygen near 8 mg/L, temperature 171, salin-
ity 35 ppm, pH near 8, and ammonium and
nitrite levels less than 1. The water was changed
3 times per week or when some parameter
was destabilised.
When the animals arrived at the aquarium,
they were separated by sex and age (juveniles
and adults) based on their size and placed on
2 different tanks. The diameter of each animal
(at the ambitus) was measured with a precision
Fig. 1. Righting behaviour. A. A sea urchin with its aboral face visible in the water column. B. A sea urchin in a half-righting
position. C. Sea urchin reaching the complete righting position. Modified from Binyon (1972).
4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)
calliper and expressed in millimeters. Indi-
viduals with diameters ranging from 16 mm
to 55 mm were used in the experiments. This
allowed us to assess the entire diameter range
of A. dufresnii in the Golfo Nuevo area, from
juveniles to adults.
Pre-test definitions: The initial position
was defined as the point at which the individu-
al’s aboral face is directed toward the substrate
and the oral face is directed toward the environ-
ment (Fig. 1A). HRB was defined as the point
at which the individual made a 90° angle with
respect to its oral plane (Pearcy, 1973) (Fig. 1B).
The moment when the individual fully rested
all of its tube feet on the substrate, returning to
its original position with the oral face toward
the substrate and the aboral face toward the
environment (Fig. 1C), was defined as CRB.
CBR and HBR tests were carried out in
tanks with aeration, controlled water qual-
ity parameters, and enough water volume to
completely cover the animals while avoiding
contact with tank walls and other individuals.
All animals were handled with extreme caution
to avoid damage to the tube feet, which could
have an impact on the final results. Each ani-
mal was positioned with its aboral face exposed
to the water column, and the time it took to
perform HRB and CRB was recorded using a
stopwatch (Fig. 2).
Experimental design:
Half-righting behaviour (HRB) vs. com-
plete righting behaviour (CRB): these behaviours
were tested on 100 adult and juvenile A. dufres-
nii specimens. To reduce individual variability
in the righting starting rate, measurements of
both HRB and CRB were initiated as soon as
the urchin was inverted in this study.
Influence of sex and diameter: The dif-
ference between sexes and the influence of
diameter were tested for CRB with different
objectives. On the one hand, to see if it was
necessary to separate the individuals in the
experiments into females and males as separate
groups, and on the other hand, to see the effect
of the diameter on the CRB.
Experiment 1- Serial turnover repetitions:
Three times in a row, the same animal was
inverted, and the HRB and CRB times were
recorded each time. In this experiment, we
were able to determine the effect of fatigue
after the effort required for righting behaviour.
Animals were kept at 9 °C (± 1 °C) during the
experiment.
Experiment 2- Thermal shock: The animals
were placed in three separate temperature-
controlled recirculation aquaculture systems
(RAS). The animals were acclimated at 9 °C
(±1 °C). The HRB and CRB were recorded
after a thermal shock at 13 °C, 16 °C, and
19 °C (animals were exposed for 24 hours),
Fig. 2. Different patterns of righting and half righting behaviour. A. initial position. B. Half righting position at 70°. C. Half
righting position at 90° (HRB). D. Complete righting position (CRB).
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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)
and the animals were gradually returned to
their original temperature after the experiment
was completed.
Experiment 3- Spawning stress: A total of
30 animals were induced to spawn by injecting
0.3 mL of KCl 0.5 M through the peristomal
membrane to see if this stressor affects CRB.
Another group of 20 animals served as controls
and received no injections. Both sets of animals
HRB and CRB righting times were recorded 24
hours after injection.
Data analysis: The experiments and the
effects of sex and diameter were analysed using
Generalised Linear Models (GLM). Because
each stressor effect was studied in different
groups of animals, each dataset obtained was
analysed using a specific model. Temperature
and post-spawning effects on CRB and HRB
were tested using diameter as a covariate. To
investigate the effects of serial turnover rep-
etition on HRB and CRB, a GLMM analy-
sis was performed (Zuur, 2009). To begin, a
graphical data exploration was performed to
better understand the relationship between
the response variable and each explanatory
variable. The amount of variance explained by
the model and the deviance (D2 complex that
includes the interaction between the factors,
indicating their dependence) were used to eval-
uate model fit to the data. Three criteria were
used to select the best model: i) The Akaike
Information Criterion (AIC), which assesses
the model’s fit to the data as well as its com-
plexity; ii) Residual analysis (observed versus
estimated residual plots); and iii) The Principle
of Parsimony (the simplest possible model).
The AIC values are included in the results. All
analyses were carried out with the help of the
free software R Studio.
RESULTS
Half righting behaviour vs. Complete right-
ing behaviour: The righting behaviours followed
a distinct pattern. Some people reached 70° and
paused for a long time before continuing to 90°,
from which they fell back down and completed
the CRB. During the righting behaviour, three
different situations were observed: 1) individu-
als reached 90° and quickly fell down without
braking, 2) individuals reached 90°, briefly
paused, and then slowly completed the right-
ing behaviour, taking approximately the same
amount of time for both phases, and 3) individ-
uals began the movement, nearly reached 90°,
and then fell back down to the initial position
(aboral face to the substrate) (Fig. 2).
Fig. 3. Half righting behaviour and Complete Righting behaviour (in seconds) of each individual with different diameters (in
mm) in adults of Arbacia dufresnii.
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There was no difference in the times for
righting and half-righting behaviour. Fig. 3
shows the paired data of each individuals right-
ing and half-righting times. The data points
formed a single cluster rather than a discernible
pattern, as shown in the figure. Furthermore,
residual analysis revealed that the HRB model
provided the poorest fit for explaining the data
(Fig. 4). Table 1 displays the GLM analysis
results along with the corresponding Akaike
values and degrees of freedom.
Influence of sex and diameter: The find-
ings reflect the wide range of sizes examined
in this study. Larger individuals took longer to
complete CRB, with values ranging from 150
to 250 seconds in animals larger than 30mm
compared to values less than 150 seconds in
animals smaller than 30mm (Fig. 3). According
Fig. 4. Analysis of residuals of the best models for Half Righting Behaviour (A) and Complete Righting Behaviour (B) in
adults of Arbacia dufresnii.
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to the GLM analysis, sex had no effect on CRB
time (Fig. 5), but diameter did (Fig. 6).
Experiment 1: Serial turn over repetitions
revealed no evidence of extenuation, with CRB
times less than 100 seconds in three repetitions.
The GLMM analysis revealed no statistically
significant differences in time between serial
repetition experiments (Fig. 7).
Experiment 2: Temperature: The model
that took into account both diameter and tem-
perature provided the best fit to the data, with
a lower AIC value. This suggests that diam-
eter and temperature had separate effects on
the righting response. According to the GLM
analysis, the temperature shock only influ-
enced CRB time at the higher temperature of
19 °C, increasing the time required to com-
plete CRB to 200 seconds. The righting time
remained constant at temperatures of 16 °C and
13 °C, with CRB times of less than 100 seconds
(Fig. 8).
Experiment 3-Post-spawning: According
to the GLM analysis, the stress caused by
spawning had no significant effect on CRB.
However, post-spawned sea urchins exhibited
greater variability in righting time with higher
CRB times, reaching 150 seconds compared to
non-spawning sea urchins who reached values
less than 75 seconds (Fig. 9). Furthermore,
spawned individuals died at a 10 % rate, where-
as non-spawned sea urchins (control group)
died at 0 %.
Fig. 5. Arbacia dufresnii. Sex influences complete righting
behaviour (CRB). Female and male righting behaviour
times in seconds without stress (N = 108). There were no
statistically significant differences between sexes.
Table 1
Generalised linear models and generalised linear mixed models analysis for sex and diameter and stressors variables in
Arbacia dufresnii.
ANALYSIS d.f. Akaike
Half righting vs. complete righting behaviour
Righting 5 976.143
Half righting 5 961.737
Sex and diameter (GLM)
Null model 2 1077.347
Righting ~ diameter + sex 4 1050.731
Serial repetitions (GLMM)
Righting ~ serial repetitions 1 1901.6
Temperature (GLM)
Null model 2 257.599
Righting ~ diameter + stress 5 239.729
Post spawning (GLM)
Null model 2 417.649
Righting ~ diameter + stress 4 392.390
For the GLMs, in bold there are the best adjustments according to the Akaike criterion.
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DISCUSSION
It is critical to have an indicator that can
be determined with a small margin of error in
order to accurately determine the stress levels
of sea urchins. Percy (1973) proposed that sea
urchins spend approximately 80 % of their
time raising themselves to a 90° angle and can
gradually or rapidly descend by releasing their
supporting tube feet to achieve the CRB. In
the current study, it was discovered that some
individuals reached approximately 70° and then
paused for a long period of time before con-
tinuing up to 90° and eventually reaching the
Fig. 6. Arbacia dufresnii. The effect of diameter on complete righting behaviour (CRB). Juveniles and adults (N = 247)
righting behaviour times in seconds.
Fig. 7. Arbacia dufresnii The effects of serial repetitions on
complete righting behaviour (CRB). Righting behaviour
times in seconds.
Fig. 8. Arbacia dufresnii. The effect of temperature on
complete righting behaviour (CRB). Righting behaviour is
measured in seconds. The 19 °C treatment was significantly
different from the 13 °C and 16 °C treatments (*denotes
significant differences with a P-value of 0.00955).