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

urchin’s 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.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)

(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).

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 individual’s 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.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)

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).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)

Complete Righting Behaviour (CRB). Three

distinct patterns of behaviour were described:

1) reaching 90° and rapidly falling without

braking, 2) reaching 90°, pausing briefly, and

then gradually descending while exerting the

same force used to reach 90°, and 3) beginning

the movement, almost reaching 90°, and then

falling back to the initial position (aboral face to

the substrate). These behaviours observed in A.

dufresnii to reach the CRB were similar to those

observed in Strongylocentrotus droebachiensis

and have previously been reported in other

equinoderms, particularly sea urchins (Percy,

1973; Romanes & Ewart, 1881).

HRB has been used as an indicator of

stress in species as Lytechinus variegatus and

Strongylocentrotus droebachiensis (Böttger et

al., 2001; Brothers & McClintock, 2015; Chal-

lener & McClintock, 2017; Hagen, 2020; Percy,

1973) as well CRB in species as Strongylocen-

trotus fragilis (Jackson, 1912), Strongylocentro-

tus purpuratus, Diadema antillarum (Philippi,

1845), Echinometra lucunter (Linnaeus, 1758)

and Strongylocentrotus intermedius (A. Agassiz,

1864) (Shi et al., 2018; Sherman, 2015; Sun et

al., 2019; Taylor et al., 2014; Giese & Farman-

farmaian, 1963). Determining the precise posi-

tion of the Half Righting Behaviour (HRB)

can be difficult, especially when the animals

complete the behaviour in less than a minute.

Establishing a clear indicator is difficult due to

the variability observed during the initial stage

of this behaviour. Furthermore, determining

the precise moment when an animal reaches a

90° angle is difficult and subjective because it

can be influenced by factors such as observer

position and water reflection. Furthermore,

as observed in this study, animals may use a

70° angle rather than a 90° angle. Given that

they did not move their spines or ambulacral

feet, this appears to be a resting position.

It is critical that the observer maintains the

same position throughout the trial in order to

accurately determine when the animal reaches

the 90° angle. The CRB, on the other hand,

provides a clear and unequivocal indicator. To

calculate the CRB time, the sea urchin must

restore its natural oral position by placing all

of its tube feet on the ground. In the CRB, the

angle or observed position is irrelevant, and

there is no doubt when sea urchins have accom-

plished it. It would be more reliable to concen-

trate efforts on determining the CRB position

rather than the HRB. Furthermore, there was

no significant variability between measures in

the current study’s treatments, indicating that

this would be a reliable indicator. However,

no significant differences between HRB and

CRB were found in this study of A. dufresnii.

Although HRB appeared to be an ambiguous

and subjective indicator that was not tested on

as many species as CRB, it is important to note

that it would be more useful in aquaculture tri-

als. It is known that in stressful situations, some

species can take up to 10 minutes to reach the

CRB (Taylor et al., 2014), making it difficult

to use the CRB to measure many animals in a

practical or rutinary manner. As a result, it is

critical to consider the type of test that will be

performed when deciding whether to use HRB

or CRB as a parameter.

The aim of this study was to define an indi-

cator based on righting behaviour, and deter-

mining whether sex and diameter had any effect

on it was critical. On the one hand, the sex had

no effect on the CRB, which was expected based

Fig. 9. Arbacia dufresnii The effect of injection spawning on

complete righting behaviour (CRB). Righting behaviour is

measured in seconds. There were no statistically significant

differences between control and spawned animals.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)

on previous research in this species. However,

it is well known that diameter influences motor

behaviour (movement) on both horizontal and

vertical surfaces (Domenici et al., 2003). In

species such as L. variegatus, S. purpuratus, and

S. droebachiensis, CRB is also influenced by

diameter, with smaller individuals completing

the behaviour faster than larger ones (Challe-

ner & McClintock, 2017; Sonnenholzner et al.,

2010; Percy, 1973). Larger individual have more

variance in CRB than the smaller ones (Chal-

lener & McClintock, 2017). However, in a study

with S. intermedius, the diameter had no effect

on the CRB (Zhang et al., 2017). In the current

study, there was a difference in CRB between

smaller and larger individuals. Larger ones (up

to 30 mm) achieved times between 150 and

250 seconds, while smaller ones achieved times

less than 150 seconds. This may be due to the

force exerted by the animals using their tube

feet (Lawrence, 1975), which appears to be pro-

portional to the individual’s size. The size of the

spines may also explain the difference in time

to reach the CRB (Sherman, 2015). Smaller

individuals in A. dufresnii had larger spines,

which could justify the effort and time required

for larger individuals to perform the CRB, as

reported by other authors such as Challener

& McClintock (2017). As a result, when using

CRB as an indicator, it is critical to consider

the individual’s size. To refine the indicator, it

may be necessary to examine the canopy (the

diameter including the spines) rather than just

the diameter to determine the CRB (Nishizaki

& Ackerman, 2007). More trials would be

conducted to measure the size of the spines in

juveniles and adults of A. dufresnii in order to

verify the information. Furthermore, CRB may

differ between species due to factors such as

seawater temperature and predator presence.

S. purpuratus, for example, took an average

of 12 seconds to perform CRB in individuals

ranging in size from 125 to 54 mm in diam-

eter, whereas A. dufresnii took an average of

45 seconds in individuals ranging in size from

16 to 50 mm in diameter. Despite these inter-

species differences, the CRB can be used in

experiments and aquaculture to evaluate the

well-being of individuals within a species under

various conditions.

Fatigue during serial repetitions of the

CRB has not been observed in sea urchins

(Kleitman, 1941; Lawrence & Cowell, 1996).

In this study, A. dufresnii did not show fatigue

when the CRB was repeated three times in

a row. The absence of fatigue makes routine

welfare assessments more practical, and it sup-

ports the hypothesis that the CRB is a good

indicator of the health and condition of animals

in captivity. Because the variation in a single

count, or three counts, is not that great, a single

test could be performed to evaluate the state

of the animals, reducing time and improving

data collection. This would also be beneficial

to include the CRB in the data collected during

routine sampling.

The temperature of the seawater can affect

the CRB by affecting metabolism and the

mechanical function of the tube feet. At low

temperatures, the tube feet stretch and move-

ment and adhesion to the substrate slow (Percy,

1973). Rosales-Schultz (2016) found that high

temperatures influenced Tetrapygus niger

(Molina, 1782) behaviour, reducing mobility

until total cessation near 32.7 °C. High temper-

atures can cause delays or failures in righting

behaviour, possibly due to heat narcosis, which

causes weakness, limpness, and contraction

of the tube feet (Percy, 1973). A. dufresnii is

a temperate species with a seawater tempera-

ture range of 0 °C to 20 °C, with 19 °C being

near the upper limit. As expected, longer CRB

times were observed at the highest seawater

temperature (19 °C) in this study. Brothers and

McClintock (2015) discovered a 50 % decrease

in the number of L. variegatus individuals capa-

ble of performing the CRB between the first

and tenth day of temperature exposure (28 °C

and 32 °C). The current study discovered two

distinct failure modes when performing the

CRB at high temperatures (19 °C), including

the inability to exceed the 30–40 ° angle at low

temperatures and remaining in a dorsal posi-

tion with continuous translational or rotational

movements at high temperatures. Furthermore,

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59015, marzo 2024 (Publicado Mar. 01, 2024)

the times of CRB at 19° were significantly dif-

ferent when compared to 13 °C and 16 °C,

with values near 150 seconds and less than 100

seconds, respectively; the lowest temperatures

did not show significant differences. To avoid

measurement errors, it is critical to consider

the influence of temperature on the CRB during

experimentation or when using this behaviour

as an indicator. Furthermore, the CRB can pro-

vide information on a species’ optimal culture

temperature because the time to perform the

CRB may be the shortest recorded when the

animal is in optimal conditions. According to

Ancin et al. (2021), 15 °C appeared to be a suit-

able temperature for culturing A. dufresnii, and

these findings would support this hypothesis

while also confirming the CRB as a reliable

indicator of animal health.

Spawning induction is commonly used

in scientific experiments with various appli-

cations in aquaculture. Sea urchin gametes

are extremely valuable due to their numerous

applications in human consumption, antioxi-

dant capacity, and biotechnology (Crespi-Abril

& Rubilar, 2021). However, there have been

few studies on how spawning stress affects sea

urchin behaviour and welfare, and there is no

evidence of this effect on A. dufresnii. In this

study, sea urchins that were spawned had high-

er variance but longer CRB times (greater than

150 seconds) than those that were not spawned

(less than 60 seconds). The high variability and

lack of significant differences observed in the

statistical analysis would be explained by the

small sample size used for this analysis (30 ani-

mals). However, there is a significant difference

between the treatments, which is supported by

a high mortality rate, indicating the stress that

these individuals have experienced. However,

because the control of this experiment was only

the absence of injection, it would be useful to

conduct additional trials with a control that

only simulated the injection (with no solution).

The effect of the injection’s stress would be

studied in this manner. As a result, the sample

size combined with the type of control would

not accurately represent the true response to

this stress, and the response may be overesti-

mated or underestimated.

The current study is the first to exam-

ine behaviour in Arbacia dufresnii in aqua-

culture systems, and it supports the use of

righting behaviour as an indicator of stress in

this species.

Ethical statement: the 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 fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

We are grateful to ERISEA S.A. for provid-

ing the space and facilities for the experiment.

This work was funded by the I+D agreement

between CONICET and EriSea S.A. The sea

urchins were collected under the authority of

Provincial Permit N°868/90.

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