Locality matters: Variation in the reproductive cycle and population structure of subtropical sea urchins

Abstract

The life cycle of many Echinoidea species remains poorly understood despite research conducted in temperate and tropical-subtropical regions. Common species in the Central-Eastern Atlantic’s shallow waters include Paracentrotus lividus, Arbacia lixula, and Sphaerechinus granularis. Nevertheless, significant gaps in understanding their life cycles persist. This study discusses the reproductive cycles of three sea urchin species in rocky coastal ecosystems around Gran Canaria Island (Spain) (27º45´N, 15º45´W) from June 2020 to May 2021. Morphological measurements reveal that test length increases without a corresponding weight gain. The average size at first maturity (L₅₀) was greater in females (A. lixula 46.26 mm; P. lividus 46.03 mm; S. granularis 49.67 mm SL) than in males (A. lixula 43.55 mm; P. lividus 42.82 mm; S. granularis 48.57 mm SL). The gonadosomatic index in females exceeded that of males. Histological analysis showed oocytes at various developmental stages, indicating asynchronous ovarian development with successive batch spawning seasons. Reproductive activity was generally observed during the warm season for all three species, likely coinciding with increased nutrient availability in Canarian waters. Notably, P. lividus was the only species to show two reproductive seasons annually in San Cristobal. DNA analysis confirmed species identification and provided new fragments of the COI gene, now available in GenBank for future population analysis. These findings represent the first reproductive data for these species in the North Atlantic region, offering valuable insights into their populations and establishing baseline information for managing sea urchin populations.

Introduction

Sea urchins are marine invertebrates that play a crucial role in controlling algae growth by limiting their abundance and distribution, primarily through grazing [1]. However, they are also known to capture suspended particles using their tube feet [2]. The distribution of sea urchins has been associated with some physical and biological factors, such as water motion, food availability, predator presence, and the availability of refuge areas [3,4]. These organisms are dioecious, with no known sexual dimorphism [2]. Regular echinoids possess five gonads distributed in the interambulacral zone, have external fertilization, and indirect development of the larvae [5]. Although the life cycles of many echinoid species remain poorly understood, particularly concerning their geographical distribution and adaptation to specific environmental conditions, research has been conducted on sea urchins in temperate and tropical-subtropical latitudes [6–11]. In the Central-Eastern Atlantic Ocean, Paracentrotus lividus (Lamarck 1816), Arbacia lixula (Linnaeus 1758), and Diadema africanum Rodríguez, Hernández, Clemente & Coppard 2013 are among the most representative species.

The distribution of several sea urchin species has been studied around the Iberian Peninsula, Mediterranean Sea, and Canary Islands [4,12–15]. Due to their high abundance and biomass, P. lividus, A. lixula, and Sphaerechinus granularis (Lamarck 1816) have received particular attention for their role as bioeroders in rocky ecosystems [16–18]. P. lividus and A. lixula are distributed along the Mediterranean Sea and North Atlantic Ocean, with their special distribution influenced by a sheltered-exposed gradient, hydrodynamic resistance, and microhabitat preference when coexisting [4,19,20]. In the Mediterranean Sea and the Atlantic, P. lividus has shown a behavior related to excavating their burrow, which fits the animal’s size, and provides protection against their predators and water movement [21]. Meanwhile, A. lixula is primarily a grazer of encrusting coralline algae and is typically found in shallow, exposed, and usually vertical rocky areas where it withstands strong water motion and turbulent conditions [4,19,20,22]. In the Canary Archipelago, both species share a similar distribution in shallow rocky reef habitats [19]. On the other hand, it is reported that S. granularis is distributed in the Mediterranean Sea to 100 m but is commonly found between 5 and 15 m depth [13,16]. This species inhabits various substrates and plays a significant role in grazing Corallinaceae-dominated areas. In Madeira and the Canary Islands (East Atlantic Ocean), S. granularis is found further from the coast (30–40 m) and at depths below 6 m [23,24]. S. granularis also displays a covering behavior, often concealing itself with small rocks, algae, or shells [13].

The populations of several sea urchin species have steadily declined in recent years due to overfishing [25–28] and mass mortality events caused by disease outbreaks [29–37]. P. lividus is considered an edible species in the Atlantic Ocean and the Mediterranean Sea [38], and in regions such as Galicia (NE Atlantic), where the annual landing has ranged between 400 and 750 tons per year since 1985 [39]. Due to the rising demand for this resource, natural populations are being harvested at higher rates, leading to declines in abundance [39]. S. granularis is less commonly harvested, but with the decline of P. lividus populations, it has become an alternative for local consumption in certain regions of the Hellenic Seas [13]. Although there is no official harvesting, data on its population status remains scarce [13]. On the other hand, A. lixula is considered a non-edible species and is not directly affected by harvesting. However, some authors suggested that the increasing A. lixula populations could impact benthic communities [15]. The loss of these sea urchin populations not only alters the structure of rocky coastal ecosystems but also reduces genetic variability, potentially affecting their adaptability to environmental changes such as temperature fluctuations, ocean acidification, and salinity shifts [40]. As a result, it is expected that natural P. lividus populations may require repopulation efforts in the future. Understanding their reproductive cycles is therefore crucial for developing conservation strategies and sustainable fishery management plans [39,41].

The reproductive cycles of these species have been documented in the Northeast Atlantic Ocean and the Mediterranean Sea, and several studies show that the breeding period depends on the locality and the season [6–11]. For example, some authors reported that near the Strait of Gibraltar (Spain), spawning occurs in different seasons depending on each site and is related to the food source in each location [8]. Information about the reproductive cycles of our target species has been reported in previous studies. A. lixula shows differences in spawning periods regarding the quantity and season in the Mediterranean Sea [9,10]. The same pattern was reported for S. granularis in the Mediterranean Sea and Madeira Archipelago (Central-east Atlantic) [7,11]. In the Canary Archipelago, information on reproduction is scarce or non-existent in species such as A. lixula or S. granularis, as most research efforts on sea urchins have focused mainly on Diadema africanum, a species whose population blooms have been considered an ecological threat with significant consequences for biodiversity and fisheries [42,43].

In this context, the present work aims to contribute to our understanding of the population dynamics of A. lixula, P. lividus, and S. granularis, the most common sea urchin species inhabiting rocky shores around the Gran Canaria Island (Central-east Atlantic, Spain) [14]. Specifically, we investigate their spatial distribution patterns and reproductive cycles across different localities. We hypothesize that these species exhibit reproductive cycle variations in response to the environmental conditions of the Canarian archipelago. To test this, we analyzed their reproductive cycles using a histological examination of oocyte development and reproductive parameters, including length frequency distributions, sex-ratios, length at maturation, and spawning seasons, to determine reproductive strategies. Additionally, DNA barcoding techniques were employed to confirm species identification and provide genetic reference data. These findings are essential for understanding the population dynamics of these species, particularly in the absence of other biological and fisheries-related estimates.

Materials and methods

1. Study area

This study was conducted in Gran Canaria Island, Spain (Central-Eastern Atlantic Ocean), 27º45´N, 15º45´W approximately, in five localities around (“Tasartico” and “La Aldea” in the West, “Arguineguín” in the South, “San Cristobal” in the East, and “Bañaderos” in the North), covering all cardinal points from June 2020 to May 2021 (Fig 1). The map was generated using geographic information system QGIS [44] and bathymetric data obtained from the GEBCO (General Bathymetric Chart of the Oceans) dataset [45].

[Figure omitted. See PDF.]

2. Field survey

At least five individuals of each sea urchin species, with different sizes, were manually collected using basic snorkeling equipment, every month during daylight, from 1 to 5 m depth at each locality. The organism collection was performed using a spatula to lever and extract the organisms. Sea urchins of different sizes, from juveniles to adults, were collected to determine the length frequency distribution and provide the length at first maturity. To avoid bias in capture, all the organisms were randomly selected in each area. After this, the animals were transported to the lab in a small case with seawater. In the laboratory, eugenol was placed in a box with seawater to anesthetize them and then euthanasia was performed by placing them in a freezer for a few minutes, to minimize and eliminate pain and distress during this process [46,47].

In addition, subsamples of sea urchins were used (5 individuals of A. lixula, 6 P. lividus, and 2 specimens of S. granularis) for DNA Barcoding analysis to confirm the ID of the organisms in this study. Tissue samples are deposited at the Senckenberg Natural History Museum Frankfurt; the DNA extractions are stored in the Senckenberg DNA Bank (Collection Numbers SMF 6949-SMF 6956). The Government of the Canary Islands allowed the collection of all the sea urchins under the permission Nº: 168/ 2020 – Tomo: 1, Libro: 401, Date: 10/06/2020.

3. Laboratory procedures

The sea urchins were weighed (Total weight, TW) with an analytic balance (Brand RADWAG, Model PS 750.R2, precision 0.001 gr), and the test height (oral-aboral axis, H), and test diameter (perpendicular to the oral-aboral axis, D) were measured with a caliper to the nearest 0.01 mm, before the dissection. Both measurements were taken in duplicate to minimize the error. Subsequently, the organisms were dissected using stainless-steel dissection scissors across the test to extract all the internal organs for analysis.

The five gonads and the gut with their content were extracted and weighed (eviscerated weight, eW). Sex was estimated from 644 samples observing the gonads macroscopically following the [6] and [15] descriptions for A. lixula and P. lividus, respectively. In the case of S. granularis, the gonad color is not representative of describing the sex macroscopically [11]. However, the state of maturity was determined for only 400 individuals through gonad histological analysis, encompassing all maturity stages, following the standard terminology for describing reproductive development [6,11,15]. Gonads were fixed with formaldehyde 4% for histological analysis to confirm the sexes. After fixation, the gonad samples were placed into cassettes and processed following the protocol of [48]. Processing of the samples included dehydration through ascending grades of alcohol, clearing in xylene, and finally imbibition in paraffin wax. Paraffin blocks were sectioned at 4 µm and sections stained with hematoxylin and eosin (H&E). The slides were mounted and examined with a light microscope (Olympus BX51TF, Japan). All the samples were analyzed in the Laboratory of Histology at the Veterinary Faculty, Universidad de Las Palmas de Gran Canaria. Moreover, the gut content was weighed to correlate the Gut Condition Index (GCI) with the reproductive cycle.

4. DNA Amplification and Sequencing

In addition, samples of the sea urchin peristomial membrane were taken and stored in absolute ethanol at −20 ºC until processed in Grunelius-Möllgaard Lab (Senckenberg Research Institute and Natural History Museum). Following the supplier’s instructions, we extracted total genomic DNA utilizing the DNeasy Tissue Kit (Qiagen). Subsequently, a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene (up to 645 bp) was amplified using the specified primers. The HCO2198/LCO1490 primer set by [49] was used for specimens of A. lixula. Genes were amplified for P. lividus and S. granularis specimens using specific echinoderm primers COIe-F/COIe-R [50]. The PCR amplification was performed in a 25 µl reaction mixture containing 21μl of ddH₂O, 1μl each of the forward and reverse primers (10μM), and 2μl of extracted DNA and the PureTaq™ Ready-to-Go™ PCR Beads (Cytiva, UK), The PCR program used the COIe-F/COIe-R primers included an initial denaturation at 95 °C/ 2 minutes, followed by 30 amplification cycles (95 °C/30 seconds, 45 °C/30 seconds, and 72 °C/1 minute), and concluded with a final step at 72 °C/8 minutes [51]. The program used for HCO2198/LCO1490 started with initial denaturation at 94°C/3 minutes, 5 amplification cycles (94°C/30 seconds, 47°C/45 seconds, 72°C/ 1 minute), then 30 cycles (94°C/30s – 52°C/45 seconds – 72°C/1 minute), and a final step at 72°C/5 minutes [49]. The reactions were carried out in an Eppendorf thermal cycler. Products that were successfully amplified underwent purification using 2μl of ExoSAP-IT PCR product cleaning reagent. The purified products were sequenced at the Senckenberg Biodiversity and Climate Research Centre (SBiK-F) and assembled with Geneious v.11.0.2 [51].

5. Ethics statement

This study involved the collection and analysis of three species of sea urchins, which are not subject to ethical approval under current Spanish legislation. According to “Real Decreto 53/2013”, of February 1, which regulates the basic standards for the protection of animals used in scientific research and education in Spain (BOE-A-2013–1337), ethical approval is only required for research involving live cephalopods and vertebrate animals. Therefore, no ethical approval was necessary for this study. In addition, no endangered or protected species were involved, and all samples were collected following local environmental regulations. All procedures complied with institutional, national, and EU regulations for the ethical treatment of marine invertebrates.

6. Data analysis

Relationships between test diameter (D), test height (H), and total wet weight (TW) of the three species were obtained to describe the population growth structure around the island. Regression of D vs. H was performed, and then a Standardized Major Axis Regression (SMA) [52] was performed using smatr package in R Studio [53] to determine if there is substantial evidence to reject this H₀ that the regression slopes between localities are equal. The regression values were then compared across localities using ANCOVA [54], performed by the emmeans package in R Studio [55]. The allometric growth of TW concerning the test diameter was estimated using a potential relationship TW = a· D^b, where a, is the intercept of the regression model, and b is the slope (or allometric coefficient). For TW versus D, the allometric coefficient of 3 indicates isometry, i.e., that sea urchins’ wet weight and diameter have a proportional growth. If the growth model is negatively allometric, the body length grows faster than the body weight. Differences between the expected value from isometric growth and values of the regression coefficient (b) were compared using a t-test [56]. This test evaluated the null hypothesis H₀: b = 3 in the TW vs. D relationship with a significance level of 5% (α = 0.05) and a critical value of t0.05, > 200 = 1.98 [56,57].

The sex ratio was calculated considering the histologically verified sexes, and to determine if the variables were independent using a Chi-square test performed with stats package in R Studio [58]. Then, a Fisher test was performed with stats package in R Studio [58], to determine if there was sex dominance across the localities. Also, to compare the size of the oocytes between species, the diameter of mature oocytes of 30 adult females (10 of each species, and 10 eggs per individual) was measured, using slides with histological images, and the cell measures were made using ImageJ software [59]. After the measurement, an ANOVA and a Tukey test were performed with stats package in R Studio [58] to compare the size of the oocytes between species. All the statistical analysis was performed using R Studio software [58].

For mature individuals (n = 187), maturity ogives, the length at first maturity (L₅₀) for both sexes, and the percentage of mature individuals by length class were calculated. The data were fitted to a normal cumulative distribution using an iterative nonlinear regression model. Additionally, a comparison of means test was conducted to assess potential differences between the L₅₀ values of both sexes. The resulting data were modeled using a sigmoid function as described below:

where P_r represents the percentage of sexually mature individuals, r is a constant indicating the slope of the curve, L₅₀ denotes the length at which 50% of individuals were mature, and L refers to the sea urchin diameter at which the P_r was calculated.

The gonadosomatic index (GSI) was calculated using the ratio of the wet weight of the five gonads to the total wet body weight of the individual to determine the spawning season (Equation 1). The Gut Condition Index (GCI) was calculated using the ratio of the wet weight of the stomach content to the total wet body weight of the individual, calculated following [60] (Equation 2).

(1)(2)

After the GSI analysis, ANOVA and a Tukey test were performed on R Studio [58] to compare the GSI values between species and localities. Moreover, an alternative approach to determine the spawning season involved analyzing the temporal variation in the frequency distribution of individuals across different stages of maturity, based on macroscopic observations of the gonads. The number of sea urchins in each maturity stage and their corresponding frequencies were computed. To estimate the Gonadosomatic Index at 50% maturity (GSI₅₀), a logistic function was applied to the proportion of mature sea urchins across 0.5 GSI intervals for both sexes, using a nonlinear least-squares regression method. The logistic equation was:

where PM_GSI is the percentage mature at GSI, a is the slope, and b is the GSI₅₀. The minimum size at maturity of males and females was taken as the smallest specimen with a GSI over GSI₅₀ based on the GSI–SL relationships [60].

In addition, dplyr [61] and tidyr [62] were used for data manipulation, and ggplot2package [63] was used for data visualization.

All data generated and analyzed during this study have been deposited in the PANGAEA repository [64], and are accessible via the following DOIs: 10.1594/PANGAEA.983883 [65] and 10.1594/PANGAEA.983888 [66].

Results

1. General characteristics of the sea urchin populations

During the sampling year, 677 sea urchins (252 of A. lixula, 359 of P. lividus, and 66 of S. granularis) were collected in the five localities described before (Fig 1), with a high frequency of organisms in the class size between 30–50 mm (Fig 2). Body measurements of sea urchins (Table 1) showed a variation in diameter for the species examined (A. lixula, 9.54 to 62.1 mm; P. lividus, 10.8 to 64.5 mm; S. granularis, 25.1 to 68 mm), where the smallest species was P. lividus (Table 1). S. granularis was the largest species (considering diameter means and height), followed by A. lixula and P. lividus. The normality of the test diameter data was assessed using the Shapiro-Wilk test, which indicated that the data were significantly non-normal, and a significant difference in diameter was observed for A. lixula and P. lividus, between the localities (Shapiro-Wilk test, F_2,300 = 36.85, p < 0.005), where the Tukey test showed that Tasartico was the locality with the smallest organisms of both species of the sampling areas (p < 0.005).

[Figure omitted. See PDF.]

[Figure omitted. See PDF.]

The regression between diameter and height was positive and significant (P < 0.05) in all the species (Fig 3), SMA results show that the relation between D and H varied across localities just in A. lixula (SMA, F_4,171 = 14.45, p = 0.005981) and P. lividus: (SMA, F_4,175 = 9.929, p = 0.005981), and there was insufficient evidence to reject the H₀ among S. granularis samples differ across localities (SMA, F_3,54 = 6.094, p = 0.1071). ANCOVA shows that Tasartico was statistically different from the other localities in P. lividus (ANCOVA, F_9,349 = 211.2, p = 2.2 × 10^-16) and A. lixula (ANCOVA, F_{9, 242} = 154.4, p = 2.2 × 10^-16).

[Figure omitted. See PDF.]

We applied the following allometric growth test diameters-to-total weight (Fig 4) equation to determine relationships in the individuals measured for each species (A. lixula: TW = 0.002 D^2.649; R² = 0.88; p < 0.001; N = 252; P. lividus: TW = 0.002 D^2.682; R² = 0.91; p < 0.001; N = 359; S. granularis: TW = 0.001 D^2.778; R² = 0.85; p < 0.001; N = 66). We observed a significant relationship between the average test diameters and total weight (Fig 4). The t-test showed that the null hypothesis of equality in the regression coefficient H₀: b = 3 was rejected for all individuals showing negative allometric growth (t-test, A. lixula: t = −10.05 < t_0.05,251 = 1.96, p = 0.0001; 95% Coefficient of Length (CL): 2.58–2.72; P. lividus: t = −16.99 < t_0.05,358 = 1.96, p = 0.0001; 95% CL: 2.64–2.72; S. granularis: t = −2.53 < t_0.05,65 = 1.99, p = 0.0001; 95% CL: 2.60–2.95).

[Figure omitted. See PDF.]

2. Reproductive cycle

2.1. Histological analysis.

A total of 353 gonad samples of sea urchins across five localities were analyzed. The sex ratio of each species was not statistically different from 1:1 (Chi-square test: A. lixula, 1:1.2 X₀² = 1.3158; N = 171; p > 0.05; P. lividus, 1:1.1 X₀² = 0.6914; N = 175; p > 0.05; S. granularis, 1:0.7 X₀² = 1.8519; N = 54; p > 0.05). The Fisher test shows that the differences between sexes across the localities were not statistically significant (p = 1; N = 353). Macroscopic observations were validated with gonad tissue histology for each species (Figs 5- 7). To describe gametogenesis and document the annual reproductive cycle, the predominant cell populations in the germinal epithelium were examined histologically (Fig 5 (A. lixula), Fig 6 (P. lividus), Fig 7 (S. granularis)). The gonadal growth pattern was divided into five stages, following descriptions by [67,68].

[Figure omitted. See PDF.]

(A) Cross section of the ovary, and (B) Cross section of testicles. The number indicates stages: 1-Gonad growing, 2-Premature, 3-Mature, 4-Spawned, and 5-Recovery. LN: Large nuclei cell, GE: Germinal epithelium, Tn: Thickened nutritive tissue, Aw: Acinar wall devoid of cells, R: remnant of mature cells. (Scale bar = 200 μm).

[Figure omitted. See PDF.]

(A) Cross section of ovary, and (B) Cross section of testicles. The number indicates stages: 1-Gonad growing, 2-Premature, 3-Mature, 4-Spawned, and 5-Recovery. LN: Large nuclei cell, GE: Germinal epithelium, Aw: Acinar wall devoid of cells, D: Densely packed mature cells, M: Mature oocyte, R: remnant of mature cells. (Scale bar = 200 μm).

[Figure omitted. See PDF.]

(A) Cross section of ovary, and (B) Cross section of testicles. The number indicates stages: 1-Gonad growing, 2-Premature, 3-Mature, 4-Spawned, and 5-Recovery. LN: Large nuclei cell, GE: Germinal epithelium, Tn: Thickened nutritive tissue, Aw: Acinar wall devoid of cells, D: Densely packed mature cells, M: Mature oocyte, R: remnant of mature cells. (Scale bar = 200 μm).

Stage I: Gonad growing. The first stage of maturity is characterized by the presence of large nuclei near the base of the germinal epithelium belonging to primitive spermatogonia or oogonia. In some cases, we could find residual oocytes or spermatozoa from the previous spawning event.

Stage II: Premature stage. This stage is characterized by mature cells, the presence of nucleated cell remnants, and thickened nutritive tissue at the edge of the ovaries or testes. In the case of the ovaries, it was common to find oocytes in different stages of maturity.

Stage III: Mature stage. This stage occurs when there is a higher proportion of densely packed mature cells (oocytes or sperm) and the nutritive tissue has considerably decreased in thickness, sometimes becoming indistinguishable.

Stage IV: Spawning. This stage is represented by large acini containing empty spaces, with numerous unspawned oocytes or spermatozoa. The acinar wall is almost devoid of any cells.

Stage V: Recovery. At this stage, the gonadal tissue is mostly depleted of reproductive cells, with remnants of mature sperm or oocytes. In some cases, the beginning of the appearance of germinal epithelium and thickening of the acinar wall can be seen.

Additionally, a significant difference in oocyte diameter during the III stage (Mature) between the 3 species (ANOVA, F_2,300 = 36.85, P < 0.001) was found. The post hoc test showed that the difference was between all the species (all the values of P < 0.001), being the oocytes of A. lixula smallest in size, and those of S. granularis being the biggest in comparison to the other two species (Table 2).

[Figure omitted. See PDF.]

2.2. Frequency of reproductive stages, length at first maturity, and gonadosomatic index.

In Fig 8, the reproductive stage frequency shows that both sexes mature synchronously. Different maturity stages were recorded each month after observing the gonad maturity of the species examined in this study (see section 3.2.1). The seasonal distribution of the five stages showed that stage II (premature) and stage III (mature) were present for almost the whole year for both sexes and in the 3 species. In A. lixula, a high frequency of immature (stage I) was recorded between January and February, stage II (premature) during March and May, stage III (mature) between June and August, stage IV between October and December, in all the localities, and both sexes. However, in P. lividus the frequency of each stage did not follow the same pattern across the localities: the highest percentage of Stage I in females appears between June and August, but in males the pattern changes with high percentages during March and June in Arguineguín and Bañaderos, just in June in El Puertito, from April to June in San Cristóbal and just in April in Tasartico. In the case of stage III, in Bañaderos and El Puertito, females and males were mature in different months across the year (females in February, and males in August and November; and females in July and males in April, respectively); in San Cristóbal, females and males are mature during August, and in Tasartico during September. Considering Stage IV, in Arguineguín we found spawned female organisms from September to February and males from December to February. In the case of Arguineguín and Bañaderos show a similar pattern, with a high frequency of female organisms in Stage IV from September to November, and males between December and February; in El Puertito, que found females in this stage from November to February; in San Cristobal, females and males show a similar pattern with a high frequency of individuals in this stage between February and March; and Tasartico females were in this stage between September and January, and males between December and March.

[Figure omitted. See PDF.]

(A) Arguineguín, (B) Bañaderos, (C) La Aldea, (D) San Cristóbal, and (E) Tasartico, by sex and species. I) Gonad growing; II) Premature; III) Mature; IV) Spawned; V) Recovery.

Finally, S. granularis shows that there were organisms in Stage I across the year in all the localities, but with a high frequency of females in January and males in February and May at Tasartico, females in July, and Males in January and May at Arguineguín, and both sexes in May in Bañaderos. Stage II was not present in the sampled organisms. In the case of Bañaderos, Stage III was present during March and August in both sexes, and in Arguineguín both sexes in different months across the year (females: August, October, February, and May; and males: June, September, October, and January). The last, Stage V, was found in samples of Tasartico, with a high frequency of organisms during January in females, and February and May in males.

The length at first maturity (L₅₀) of sea urchins around Gran Canaria Island showed that A. lixula reached the L₅₀ at 43.55 mm in males and 45.26 mm in females (Fig 9). L₅₀ was estimated for P. lividus in 42.82 mm for males and 46.03 mm for females, while for S. granularis, L₅₀ values were estimated to be 48.57 mm and 49.67 mm for males and females, respectively.

[Figure omitted. See PDF.]

The GSI generally varied between species, localities, and months as expected (Fig 10). A. lixula showed the highest values of GSI in October in San Cristobal and Tasartico, in August in El Puertito and Arguineguín, and in February in Bañaderos. The values of GSI for P. lividus showed two peaks during March and October/November (Bañaderos, San Cristóbal, and Arguineguín). However, for the rest of the localities, we observed one reproductive season in P. lividus (November in Tasartico; May to July in El Puertito). During the year sampled, S. granularis was better represented in “Bañaderos” and “Arguineguín” localities, with higher values of GSI during the summer season (August) (Fig 10). We found a significant difference in GSI values in A. lixula and P. lividus, between localities (F_7,677 = 3.186, p < 0.05). The post hoc test (Tukey test) showed that A. lixula GSI values were different in “San Cristóbal” than “Tasartico” and “Arguineguín”, and for P. lividus GSI values were different in “San Cristóbal” than the rest of the localities, and between “Tasartico” and “La Aldea” (all the values of p < 0.05).

[Figure omitted. See PDF.]

Based on the logistic function, the global GSI₅₀ values were 6.04, 4.67, and 4.34 for A. lixula, P. lividus, and S. granularis, respectively (Fig 11). For males and females, the GSI₅₀ for A. lixula was 4.24 and 5.21, for P. lividus was 1.74 and 3.61, and S. granularis was 3.08 and 3.91, respectively.

[Figure omitted. See PDF.]

3. DNA barcoding analysis

We obtained approximately 663 bp of COI sequencing, 5 individuals of P. lividus, and 2 individuals of S. granularis, except for 1 individual of A. lixula from the Gran Canaria populations. All sequences have been deposited in the GenBank database (Access numbers PQ722060-PQ722067).

Discussion

1. General characteristics of the sea urchin populations

According to morphometric measurements, the maximum diameter recorded for A. lixula (62.10 mm) in our study is comparable to other localities such as the Algerian west coast, Mediterranean Sea (63.10 mm) [10], Pagasitikos Gulf, Greece (54.66 mm) [15]. In the case of P. lividus the maximum diameter (64.50 mm) was similar to other studies, such as the one reported by [69] with a maximum diameter of 69.5 mm, on the Moroccan coast; in the case of S. granularis the maximum diameter (68 mm) was smaller than the reported in other areas such as Madeira, Portugal (90 mm) [11].

In addition, it is important to highlight that we found significant differences between the localities in test diameter just in A. lixula and P. lividus, and the locality different from the others was Tasartico, with the smaller organisms. Additionally, the relationship between the diameter and height followed a similar pattern across all three species, showing a significant positive relation between both variables. Regarding morphometric analysis, we found significant negative allometric relationships, which coincide with the research of [15] with A. lixula and [70] with P. lividus. The high standard deviation values in total weight can be explained by the fact that the sea urchins were in different stages of maturity each month during the sampling campaigns.

Although the 3 species share the same microhabitat in the sampling sites, some works explain that these species can use different resources, even in barren habitats with severe food shortages. For example, [71] reports that A. lixula consumes principally encrusting coralline algae (more than 70%), while P. lividus prefers fleshy algae (more than 60%). In the case of S. granularis, it is a species that consumes mostly encrusted algae [16], and in some cases can consume dead leaves of Posidonia oceanica with their epiphyte organisms when they are accessible [72]. Also, the characteristics of each site regarding the current, temperature, water movement, food availability, and anthropogenic influence may affect the presence of each species along Gran Canaria Island. Considering this, the low density of S. granularis close to the coast could be explained because of the high effect of water movement in all the localities, and the collection method used. [24] described this species as well distributed in calm areas as deeper zones on Madeira Island. This explains why we found them easily in the quietest areas such as the natural pools of Bañaderos and Arguineguín.

2. Reproductive cycle

2.1. Histological analysis.

The histological analysis of female gonads of sea urchin species showed oocytes in different developmental states, indicating that this species has asynchronous ovarian development with successive batch spawner seasons, as observed in P. lividus from other latitudes [6,69,73]. In addition, in Gran Canaria Island, males and females of P. lividus mature simultaneously, which agrees with the results of [60] who worked with the same species on the Atlantic coasts of France.

2.2. Frequency of reproductive stages, length at first maturity, and gonadosomatic index.

Our results of reproductive seasons in A. lixula showed a variation according to the localities, mainly concentrated in autumn (in Bañaderos, San Cristobal, and Arguineguín in September, in Tasartico and La Aldea in November). This result differs from the report of [9] with a reproductive season between May and July in the Mediterranean Sea. Furthermore, in our study, we highlighted 2 reproductive peaks (with GSI values higher than the GCI) only in A. lixula in San Cristóbal and Tasartico, and P. lividus in San Cristóbal, with the highest values between summer and autumn, and the second and lowest between winter and spring (Fig 9). Recently, [10] also found two spawning periods for A. lixula: massive between spring and summer and less intense during autumn in the southwestern Mediterranean. In the case of A. lixula, GSI values were higher when the sea temperature was higher too, showing the strong relationship between both variables [20].

Our study demonstrates that the reproductive cycle of P. lividus varies depending on the seasons and locations. The maturity stage (III) aligns with late summer to early autumn, consistent with findings in the Cantabrian Sea [73]. Regional differences include late spring/early summer maturation on the French coast [60], and the peak of spawning in spring to early summer in the northeastern Spanish Mediterranean [74]. In Sardinia, P. lividus shows a primary spring spawning period [75], similar to our observations.

Considering the Canary Archipelago, [76] reported that P. lividus shows the main reproductive peak during August, October, and December; [77] reported several maturity seasons throughout the year in Tenerife Island, although the main period was between September and February. The maximum values of GSI were reported during summer, which coincides with our results in Gran Canaria.

In contrast, [75] reported two spawning events for P. lividus in a Marine Protected Area of Sardinia (June and March) with less anthropogenic pressure, suggesting a successful gamete production compared to a high-pressure zone, where only one spawning event occurred, and fertile individuals were less abundant. Smaller individuals in high-pressure zones matured earlier, while larger individuals in low-pressure zones contributed more to reproduction due to their capacity for larger gonads and higher gamete output. Similarly, in Bañaderos and Arguineguín, high anthropogenic pressure was associated with lower GSI values.

In the case of S. granularis, [11] reported that the GSI had higher values in November, with two spawning peaks: one stronger in November and one weaker in April, in Madeira Island. However, [13] report that the major spawning event of S. granularis in the Aegean Sea occurs in spring, and then (with no histological corroboration) a second minor spawning event in early autumn (September), and [78] report two spawning seasons in the Algerian coasts: first during spring and the highest peak in autumn. In contrast, [79] in Western Brittany shows that a reproductive peak was marked by a decrease in the GCI and an increase in the water temperature during summer (June to August). Our results are consistent with those described by [79] and show higher GSI values in all the sampling sites from August to September.

One explanation we found regarding the differences in the reproductive cycle among localities is the process explained by [6], which shows that the differences in food availability to the population’s habitats may lead to a variation in their nutritional conditions. In addition, [80] research on Echinus esculentus on Scotland’s coasts, provides information about how during winter months when the nutrient concentration increases in the water column induces a rapid development of gonadal mass, consequently, the quality and availability of the nutrients affect the GSI, and then cause the reproductive cycle to vary.

Another explanation was suggested by [81], who hypothesized that spawning in P. lividus is linked to seasonal phytoplankton blooms. In the case of other species of sea urchin, Echinometra lucunter, in the Caribbean Sea, the rise of the temperature stimulates the gonad development and the increase of the phytoplankton because the coastal upwelling triggers partial or full spawning in both sexes at the same time [82]. In the Canary Archipelago, several oceanographic processes affect phytoplankton development, such as the seasonal variation of the currents, the eddies, and the wind. According to the research of [83], the maximum wind speed in Gran Canaria Island was during the summer season, and this pattern is highly related to the presence of the eddies, which seems to increase the primary production because of the upwelling of deep nutrient-rich water into the euphotic layer in oligotrophic waters. In the case of the southwest of Gran Canaria, during the summer season, specifically in June, there is a prevalence of cyclonic eddies, with an increase of nutrients causing an increase of the phytoplankton. Additionally, they reported that the phytoplankton increased during autumn or the beginning of the winter season. Our results show that the peak of the reproductive season in almost all the areas around Gran Canaria Island was related to these two seasons: high during summer and lower during autumn, which could be closely associated with spawning in the season where sea urchin larvae can find high concentrations of food for their development.

There is scarce information about the sexual maturity of these species in the Central-Eastern Atlantic, specifically in the Canary Islands area. This knowledge gap highlights the importance of our results, collected over one year in five different localities for the three most abundant species in the area. In this sense, [84] and [85] described the sexual maturity of A. lixula on the Adriatic Sea and P. lividus on Tenerife Island, respectively. These authors found a diameter of sexual maturity for A. lixula (between 9.2 and 14 mm) and P. lividus (between 10.4 and 14.5 mm) lower than our results. In addition, [82] reported that the P. lividus collected were mature with a diameter test between 36–60 mm, and [77] reported an L₅₀ (first maturity) of 26.12 mm, and L₉₅ (mass maturity) at 40.56 mm in Tenerife Island. Our results show that the size of the first maturity in P. lividus in Gran Canaria Island is higher than in other localities around the world (42.82 mm for males and 46.03 mm for females). This pattern may be explained by reduced fishing pressure in the area, as this species is not commonly used as bait in Gran Canaria. Furthermore, its tendency to inhabit small cavities likely makes it less accessible to fishers, which may contribute to the presence of larger individuals. In the case of the other species, there are no records of the size of the first maturity in the Canarian Archipelago, for that reason, this work provides new information to understand the populations in this North Atlantic region, and to set a baseline data for management plans for sea urchin coastal populations.

3. DNA barcoding analysis

The COI sequences obtained in our study for P. lividus, A. lixula, and S. granularis showed more than 95% similarity with existing sequences in GenBank. For P. lividus, the sequences had between 99.54% and 99.85% similarity with over 100 GenBank sequences, including those reported by [47] (Access numbers AY630792 to AY630918), confirming species identification. Similarly, A. lixula sequences exhibited 99.04% and 100% similarity with more than 100 GenBank sequences, including those reported by [86] (Access numbers JQ745096 to JQ745256), further validating the identification. For S. granularis, the sequence reported by [87] was 96.61% similar to ours. However, the COI region analyzed in this study differed from that of [87]. Specifically, in S. granularis sampled from the Canary Islands, the primer COIa (5’-AGT ATA AGC GTC TGG GTA GTC-3’) was used, which amplified the region from 540 bp to 683 bp. We used the primer COIe described by [47] (5’- ATA ATG ATA GGA GGR TTT GG-3’), one of the most common primers for echinoderm sequences used worldwide, which amplifies from 1 bp to 680. Therefore, only 140 bp (26% of the sequence) overlapped between our S. granularis COI sequences and those amplified by [87] (Access numbers AY183288 to AY183290). Our sequences represent new fragments of the COI gene sequenced for the 3 species and available in GenBank for future studies and metabarcoding efforts.

Conclusions

Morphological measurements of sea urchins showed a significant increase in length without a proportional gain in weight, because the weight of each organism is intimately linked to its reproductive state, given that the gonads play a significant role in the variation of the animal’s weight. Sea urchin species displayed oocytes at different developmental stages, indicating asynchronous ovarian development with successive batch spawner seasons. The highest reproductive peak generally occurs during the warm season for all three species examined in this study, which could coincide with increased nutrient availability in the Canarian Archipelago. Specifically, P. lividus was the only species that showed two reproductive seasons in San Cristobal annually. The environmental conditions at each site seem to be the main factors influencing the differences found in the reproduction of sea urchins. Furthermore, females of our three species were mature at a larger size than males, which could contribute to the greater biomass of mature females than males and to maximizing the egg-producing biomass. In addition, the accessibility of the localities and the anthropogenic pressure (e.g., pollution, fisheries) may impact population structure and size of first maturity, consequently affecting the development of these species. The information provided in this study will offer a baseline about the population biology of sea urchin species. It will help to create a plan for fishery management along the North East Atlantic region, especially in the Canarian Archipelago, considering the real value of the minimum harvestable size of the organisms, maximum catch per capita, minimizing the pressure over this resource and protecting it for future generations.

Acknowledgments

The authors thank the Government of the Canary Islands for providing the scientific permit to collect the samples for this study. Raibel Nuñez-Gonzalez thanks the Senckenberg Museum of Natural History and Grunelius-Möllgaard Lab technicians for helping during the DNA sample analysis. Thanks to the people who helped us during the sampling campaigns. Finally, we greatly thank the anonymous reviewers, whose comments improved the quality of this manuscript.

References

1. 1. Valentine JF, Heck KL Jr. The role of sea urchin grazing in regulating subtropical seagrass meadows: evidence from field manipulations in the northern Gulf of Mexico. Journal of Experimental Marine Biology and Ecology. 1991;154(2):215–30.

* View Article

* Google Scholar

2. 2. Ocaña A, Pérez-Ruzafa A. Los equinodermos de las costas andaluzas. Acta Granatense. 2004;3:83–136.

* View Article

* Google Scholar

3. 3. Carpenter RC. Predator and population density control of homing behavior in the Caribbean echinoid Diadema antillarum. Mar Biol. 1984;82(1):101–8.

* View Article

* Google Scholar

4. 4. Bulleri F, Benedetti-Cecchi L, Cinelli F. Grazing by the sea urchins Arbacia lixula L. and Paracentrotus lividus Lam. in the Northwest Mediterranean. Journal of Experimental Marine Biology and Ecology. 1999;241(1):81–95.

* View Article

* Google Scholar

5. 5. Pérez SA, Gil DG, Rubilar Panasiuk CT. Echinodermata. In: Calcagno JA, Editor. Los invertebrados marinos. Buenos Aires: Fundación de Historia Natural Félix de Azara Press. 2014. p. 295–316.

6. 6. Byrne M. Annual reproductive cycles of the commercial sea urchin Paracentrotus lividus from an exposed intertidal and a sheltered subtidal habitat on the west coast of Ireland. Mar Biol. 1990;104:275--89.

* View Article

* Google Scholar

7. 7. Guillou M, Lumingas LJL. The reproductive cycle of the ‘blunt’ sea urchin. Aquaculture International. 1998;6(2):147–60.

* View Article

* Google Scholar

8. 8. Sánchez-España AI, Martínez-Pita I, García FJ. Gonadal growth and reproduction in the commercial sea urchin Paracentrotus lividus (Lamarck, 1816) (Echinodermata: Echinoidea) from southern Spain. Hydrobiologia. 2004;519(1–3):61–72.

* View Article

* Google Scholar

9. 9. Wangensteen OS, Turon X, Casso M, Palacín C. The reproductive cycle of the sea urchin Arbacia lixula in northwest Mediterranean: potential influence of temperature and photoperiod. Mar Biol. 2013;160(12):3157–68.

* View Article

* Google Scholar

10. 10. Elakkermi M, Mezali K, Soualili DL. Interpopulation variability of the reproductive cycle of Arbacia lixula (Echinodermata: Echinoidea) in the Mostaganem shallow-water area (south-western Mediterranean). Regional Studies in Marine Science. 2021;45:101810.

* View Article

* Google Scholar

11. 11. Lourenço S, José R, Neves P, Góis A, Cordeiro N, Andrade C, et al. Population Density, Reproduction Cycle and Nutritional Value of Sphaerechinus granularis (Echinodermata: Echinoidea) in an Oceanic Insular Ecosystem. Front Mar Sci. 2022;8.

* View Article

* Google Scholar

12. 12. Sala E, Zabala M. Fish predation and the structure of the sea urchin Paracentrotus lividus populations in the NW Mediterranean. Mar Ecol Prog Ser. 1996;140:71–81.

* View Article

* Google Scholar

13. 13. Vafidis D, Antoniadou C, Ioannidi V. Population Density, Size Structure, and Reproductive Cycle of the Comestible Sea Urchin Sphaerechinus granularis (Echinodermata: Echinoidea) in the Pagasitikos Gulf (Aegean Sea). Animals (Basel). 2020;10(9):1506. pmid:32858926

* View Article

* PubMed/NCBI

* Google Scholar

14. 14. Hernández JC, Clemente S, Tuya F, Pérez-Ruzafa A, Sangil C, Moro-Abad L, et al. Echinoderms of the Canary Islands, Spain. Echinoderm Research and Diversity in Latin America. Springer Berlin Heidelberg. 2012. p. 471–510.

* View Article

* Google Scholar

15. 15. Klaoudatos D, Tziantziou L, Lolas A, Neofitou N, Vafidis D. Population Characteristics of the Upper Infralittoral Sea Urchin Arbacia lixula (Linnaeus, 1758) in Eastern Mediterranean (Central Greece): An Indicator Species for Coastal Water Quality. JMSE. 2022;10(3):395.

* View Article

* Google Scholar

16. 16. Sartoretto S, Francour P. Quantification of Bioerosion by Sphaerechinus Granularis on ‘Coralugène’ Concretions of the Western Mediterranean. J Mar Biol Ass. 1997;77(2):565–8.

* View Article

* Google Scholar

17. 17. Guidetti P, Fraschetti S, Terlizzi A, Boero F. Distribution patterns of sea urchins and barrens in shallow Mediterranean rocky reefs impacted by the illegal fishery of the rock-boring mollusc Lithophaga lithophaga. Marine Biology. 2003;143(6):1135–42.

* View Article

* Google Scholar

18. 18. Bonaviri C, Vega Fernández T, Fanelli G, Badalamenti F, Gianguzza P. Leading role of the sea urchin Arbacia lixula in maintaining the barren state in southwestern Mediterranean. Mar Biol. 2011;158(11):2505–13.

* View Article

* Google Scholar

19. 19. Tuya F, Cisneros-Aguirre J, Ortega-Borges L, Haroun RJ. Bathymetric segregation of sea urchins on reefs of the Canarian Archipelago: Role of flow-induced forces. Estuarine, Coastal and Shelf Science. 2007;73(3–4):481–8.

* View Article

* Google Scholar

20. 20. Gianguzza P, Bonaviri C. Arbacia. Developments in Aquaculture and Fisheries Science. Elsevier. 2013. p. 275–83.

* View Article

* Google Scholar

21. 21. Boudouresque CF, Verlaque M. Chapter 13 Ecology of Paracentrotus lividus. Developments in Aquaculture and Fisheries Science. Elsevier. 2007. p. 243–85.

* View Article

* Google Scholar

22. 22. Chelazzi G, Serra G, Bucciarelli G. Zonal recovery after experimental displacement in two sea urchins co-occurring in the Mediterranean. Journal of Experimental Marine Biology and Ecology. 1997;212(1):1–7.

* View Article

* Google Scholar

23. 23. Pérez-Sánchez JM, Moreno-Batet E. Invertebrados marinos de Canarias. Las Palmas de Gran Canaria: Ediciones del Cabildo Insular de Gran Canaria. 1991.

24. 24. Alves FMA, Chícharo LM, Serrao E, Abreu AD. Algal cover and sea urchin spatial distribution at Madeira Island (NE Atlantic). Sci mar. 2001;65(4):383–92.

* View Article

* Google Scholar

25. 25. Sloan NA, Clark AM, Taylor JD. The echinoderms of Aldabra and their habitats. British Museum (Natural History). 1979.

26. 26. Scheibling RE, Mladenov PV. The decline of the sea urchin, Tripneustes ventricosus, fishery of Barbados: a survey of fishermen and consumers. Mar Fish Rev. 1987;49(3):62–9.

* View Article

* Google Scholar

27. 27. Guidetti P, Terlizzi A, Boero F. Effects of the edible sea urchin, Paracentrotus lividus, fishery along the Apulian rocky coast (SE Italy, Mediterranean Sea). Fisheries Research. 2004;66(2–3):287–97.

* View Article

* Google Scholar

28. 28. Bertocci I, Dominguez R, Machado I, Freitas C, Domínguez Godino J, Sousa-Pinto I, et al. Multiple effects of harvesting on populations of the purple sea urchin paracentrotus lividus in north Portugal. Fisheries Research. 2014;150:60–5.

* View Article

* Google Scholar

29. 29. Lessios HA. Population dynamics of Diadema antillarum (Echinodermata: Echinoidea) following mass mortality in Panama. Mar Biol. 1988;99(4):515–26.

* View Article

* Google Scholar

30. 30. Jones GM, Hebda AJ, Scheibling RE, Miller RJ. Histopathology of the disease causing mass mortality of sea urchins (Strongylocentrotus droebachiensis) in Nova Scotia. J Invertebr Pathol. 1985;45(3):260–71. pmid:3998481

* View Article

* PubMed/NCBI

* Google Scholar

31. 31. Dyková I, Lorenzo-Morales J, Kostka M, Valladares B, Pecková H. Neoparamoeba branchiphila infections in moribund sea urchins Diadema aff. antillarum in Tenerife, Canary Islands, Spain. Dis Aquat Organ. 2011;95(3):225–31. pmid:21932534

* View Article

* PubMed/NCBI

* Google Scholar

32. 32. Clemente S, Lorenzo-Morales J, Mendoza J, López C, Sangil C, Alves F, et al. Sea urchin Diadema africanum mass mortality in the subtropical eastern Atlantic: role of waterborne bacteria in a warming ocean. Mar Ecol Prog Ser. 2014;506:1–14.

* View Article

* Google Scholar

33. 33. Girard D, Clemente S, Toledo‐Guedes K, Brito A, Hernández JC. A mass mortality of subtropical intertidal populations of the sea urchin Paracentrotus lividus: analysis of potential links with environmental conditions. Marine Ecology. 2011;33(3):377–85.

* View Article

* Google Scholar

34. 34. Gizzi F, Jiménez J, Schäfer S, Castro N, Costa S, Lourenço S, et al. Before and after a disease outbreak: Tracking a keystone species recovery from a mass mortality event. Mar Environ Res. 2020;156:104905. pmid:32174333

* View Article

* PubMed/NCBI

* Google Scholar

35. 35. Hernández JC, Sangil C, Lorenzo-Morales J. Uncommon southwest swells trigger sea urchin disease outbreaks in Eastern Atlantic archipelagos. Ecol Evol. 2020;10(15):7963–70. pmid:32788953

* View Article

* PubMed/NCBI

* Google Scholar

36. 36. Salazar-Forero CE, Reyes-Batlle M, González-Delgado S, Lorenzo-Morales J, Hernández JC. Influence of Winter Storms on the Sea Urchin Pathogen Assemblages. Front Mar Sci. 2022;9.

* View Article

* Google Scholar

37. 37. Núñez-González R, Caballero MJ, Padilla D, Martín Barrasa JL, Castro Hernández JJ. Study of histopathology on Arbacia lixula (Arbaciidae: Arbacioida) and Paracentrotus lividus (Parachinidae: Camarodonta) with bald sea urchin disease symptoms in Gran Canaria Island, Spain. Rev Biol Trop. 2024;72(S1):e58993.

* View Article

* Google Scholar

38. 38. Bertocci I, Blanco A, Franco JN, Fernández-Boo S, Arenas F. Short-term variation of abundance of the purple sea urchin, Paracentrotus lividus (Lamarck, 1816), subject to harvesting in northern Portugal. Mar Environ Res. 2018;141:247–54. pmid:30249457

* View Article

* PubMed/NCBI

* Google Scholar

39. 39. Rey-Méndez M, Tourón N, Rodríguez-Castro B, Rama-Villar A, Fernández-Silva I, González N, et al. Crecimiento y mejora del índice gonadal en el cultivo del erizo de mar Paracentrotus lividus (Echinoida: Echinidae) en Galicia (España). Rev Biol Trop. 2015;63:261–72.

* View Article

* Google Scholar

40. 40. Tourón N, Fernández-Silva I, Rodríguez-Castro J, González NE, Catoira JL, Rey-Méndez M. Genetic Characterization ofParacentrotus lividus(Lamarck, 1816) Populations from Nw Spain and the Canary Islands. Journal of Shellfish Research. 2018;37(3):651–8.

* View Article

* Google Scholar

41. 41. Fernández-Boán M, Fernández L, Freire J. History and management strategies of the sea urchin Paracentrotus lividus fishery in Galicia (NW Spain). Ocean & Coastal Management. 2012;69:265–72.

* View Article

* Google Scholar

42. 42. Tuya F, Sanchez-Jerez P, Haroun R. Influence of fishing and functional group of algae on sea urchin control of algal communities in the eastern Atlantic. Mar Ecol Prog Ser. 2005;287:255–60.

* View Article

* Google Scholar

43. 43. Clemente S, Hernández JC, Brito A. Evidence of the top–down role of predators in structuring sublittoral rocky-reef communities in a Marine Protected Area and nearby areas of the Canary Islands. ICES Journal of Marine Science. 2008;66(1):64–71.

* View Article

* Google Scholar

44. 44. QGIS Development Team. QGIS Geographic Information System (Version 3.14 “Pi”). Open Source Geospatial Foundation Project. 2020. Available from: http://www.qgis.org

45. 45. GEBCO Compilation Group. GEBCO 2024 Grid. 2024. Available from: https://doi.org/10.5285/38a552b8-2c35-472f-e053-6c86abc06b44

46. 46. Cooper JE. Anesthesia, analgesia, and euthanasia of invertebrates. ILAR J. 2011;52(2):196–204. pmid:21709312

* View Article

* PubMed/NCBI

* Google Scholar

47. 47. Wahltinez SJ, Stacy NI, Hadfield CA, Harms CA, Lewbart GA, Newton AL, et al. Perspective: Opportunities for advancing aquatic invertebrate welfare. Front Vet Sci. 2022;9:973376. pmid:36458054

* View Article

* PubMed/NCBI

* Google Scholar

48. 48. Bancroft JD, Layton C, Suvarna SK. Bancroft’s Theory and Practice of Histological Techniques. New York: Churchill Livingstone. Elsevier. 2013.

49. 49. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3(5):294–9. pmid:7881515

* View Article

* PubMed/NCBI

* Google Scholar

50. 50. Duran S, Palacín C, Becerro MA, Turon X, Giribet G. Genetic diversity and population structure of the commercially harvested sea urchin Paracentrotus lividus (Echinodermata, Echinoidea). Mol Ecol. 2004;13(11):3317–28. pmid:15487992

* View Article

* PubMed/NCBI

* Google Scholar

51. 51. Geneious v.11.0.2. Biomatters. 2023. Available from: https://www.geneious.com

52. 52. Legendre P. Model II regression user’s guide, R edition. 14. 1998.

53. 53. Warton DI, Duursma RA, Falster DS, Taskinen S. smatr 3 - an R package for estimation and inference about allometric lines. Methods in Ecology and Evolution. 2012;3:257–9.

* View Article

* Google Scholar

54. 54. Selden R, Johnson AS, Ellers O. Waterborne cues from crabs induce thicker skeletons, smaller gonads and size-specific changes in growth rate in sea urchins. Mar Biol. 2009;156(5):1057–71. pmid:24489404

* View Article

* PubMed/NCBI

* Google Scholar

55. 55. Lenth R. Emmeans: Estimated Marginal Means, aka Least-Squares Means. 2025.

56. 56. Sachs L. Applied Statistics: A Handbook of Techniques. New York: Springer-Verlag. 1982.

57. 57. Sokal R, Rohlf F. Biometry, the principles and practice of statistics in biological research. 4th ed. New York: Freeman and Co. 2012.

58. 58. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. 2023.

59. 59. Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 2017;18(1):529. pmid:29187165

* View Article

* PubMed/NCBI

* Google Scholar

60. 60. Spirlet C, Grosjean P, Jangoux M. Reproductive cycle of the echinoidParacentrotus lividus: analysis by means of the maturity index. Invertebrate Reproduction & Development. 1998;34(1):69–81.

* View Article

* Google Scholar

61. 61. Wickham H, François R, Henry L, Müller K, Vaughan D. dplyr: A Grammar of Data Manipulation.

62. 62. Wickham H, Vaughan D, Girlich M. tidyr: Tidy Messy Data. 2024.

63. 63. Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. 2016.

64. 64. Felden J, Möller L, Schindler U, Huber R, Schumacher S, Koppe R, et al. PANGAEA - Data Publisher for Earth & Environmental Science. Sci Data. 2023;10(1):347. pmid:37268655

* View Article

* PubMed/NCBI

* Google Scholar

65. 65. Núñez-González R, Sarmiento-Lezcano AN, Tilic E, Caballero Cansino MJ, Castro Hernández JJ. Biometric measurements of dominant subtropical sea urchins in the northeast Atlantic Ocean (June 2020–May 2021). PANGAEA. 2025.

* View Article

* Google Scholar

66. 66. Núñez-González R, Sarmiento-Lezcano AN, Tilic E, Caballero Cansino MJ, Castro Hernández JJ. Oocyte dimensions of dominant subtropical sea urchins in the Northeast Atlantic Ocean (June 2020–May 2021). PANGAEA. 2025.

* View Article

* Google Scholar

67. 67. Walker CW, Unuma T, McGinn NA, Harrington LM, Lesser MP. Reproduction of sea urchins. Developments in Aquaculture and Fisheries Science. Elsevier. 2001. p. 5–26.

* View Article

* Google Scholar

68. 68. Walker CW, Lesser MP, Unuma T. Gametogenesis in regular sea urchins: Structural, functional, and molecular/genomic biology. Developments in Aquaculture and Fisheries Science. Elsevier. 2020. p. 29–50.

* View Article

* Google Scholar

69. 69. Khaili A. Reproductive cycle of the sea urchin Paracentrotus lividus (Lamarck, 1816) from the Moroccan western Mediterranean Sea: histology, gonadal index and size at first sexual maturity. Egypt J of Aquatic Biolo and Fish. 2023;27(2):745–71.

* View Article

* Google Scholar

70. 70. Herrero Barrencua A. Aspectos reproductivos del erizo común (Paracentrotus lividus) en aguas del Este de Gran Canaria. Universidad de Las Palmas de Gran Canaria. 2008. https://accedacris.ulpgc.es/bitstream/10553/11652/2/0697866_00000_0000.pdf

71. 71. Privitera D, Chiantore M, Mangialajo L, Glavic N, Kozul W, Cattaneo-Vietti R. Inter- and intra-specific competition between Paracentrotus lividus and Arbacia lixula in resource-limited barren areas. Journal of Sea Research. 2008;60(3):184–92.

* View Article

* Google Scholar

72. 72. Verlaque M. Preliminary data on some Posidonia feeders. Rapp PV Réun Commiss Internatl Explor Sci Méditerr. 1981;27(2):201–2.

* View Article

* Google Scholar

73. 73. González-Irusta JM, Goñi de Cerio F, Canteras JC. Reproductive cycle of the sea urchinParacentrotus lividusin the Cantabrian Sea (northern Spain): environmental effects. J Mar Biol Ass. 2009;90(4):699–709.

* View Article

* Google Scholar

74. 74. Lozano J, Galera J, López S, Turon X, Palacin C. Biological cycles and recruitment of Paracentrotus lividus (Echinodermata: Echinoidea) in two contrasting habitats. Mar Ecol Prog Ser. 1995;122:179–91.

* View Article

* Google Scholar

75. 75. Loi B, Guala I, Pires da Silva R, Brundu G, Baroli M, Farina S. Hard time to be parents? Sea urchin fishery shifts potential reproductive contribution of population onto the shoulders of the young adults. PeerJ. 2017;5:e3067. pmid:28289567

* View Article

* PubMed/NCBI

* Google Scholar

76. 76. González-Henríquez N, Rey-Méndez M, Catoira Gómez JL, Ruiz de la Rosa M, Louzara Fernández G, Girard Berard D. Evolución temporal del índice gonadal de los erizos marinos Paracentrotus lividus (Echinoida: Echinidae) y Diadema africanum (Diadematoida: Diadematidae) en las islas Canarias (España). Rev Biol Trop. 2015;63:251–60. http://dx.doi.og/10.15517/rbt.v63i2.23160

* View Article

* Google Scholar

77. 77. Girard Berard D. Dinámica poblacional del erizo de mar Paracentrotus lividus (Lamarck, 1816) en las islas Canarias: estudio de la biología, ecología y valoración del estado de las poblaciones. Universidad de La Laguna. 2022.

78. 78. Soualili DL, Guillou M, Semroud R. Age and growth of the echinoid Sphaerechinus granularis from the Algerian coast. J Mar Biol Ass. 1999;79(6):1139–40.

* View Article

* Google Scholar

79. 79. Guillou M, Michel C. The influence of environmental factors on the growth of Sphaerechinus granularis (Lamarck) (Echinodermata: Echinoidea). Journal of Experimental Marine Biology and Ecology. 1994;178(1):97–111.

* View Article

* Google Scholar

80. 80. Kelly MS, Owen PV, Pantazis P. The commercial potential of the common sea urchin Echinus esculentus from the west coast of Scotland. Hydrobiologia. 2001;465(1–3):85–94.

* View Article

* Google Scholar

81. 81. Starr M, Himmelman JH, Therriault J-C. Environmental Control of Green Sea Urchin,Strongylocentrotus droebachiensis, Spawning in the St. Lawrence Estuary. Can J Fish Aquat Sci. 1993;50(5):894–901.

* View Article

* Google Scholar

82. 82. Reyes-Luján J, Zapata-Vívenes É, Arrieche D, Lodeiros C. Efecto de la estacionalidad sobre el desarrollo gonadal y la composición bioquímica del erizo de mar Echinometra lucunter lucunter (Echinodermata: Echinometridae) en el Islote Lobos, Venezuela. Gayana. 2024;88(1):44–56.

* View Article

* Google Scholar

83. 83. Arístegui J, Tett P, Hernández-Guerra A, Basterretxea G, Montero MF, Wild K, et al. The influence of island-generated eddies on chlorophyll distribution: a study of mesoscale variation around Gran Canaria. Deep Sea Research Part I: Oceanographic Research Papers. 1997;44(1):71–96.

* View Article

* Google Scholar

84. 84. Ernst G, Hähnel von W, Seibertz E. Aktuopaläontologie und Merkmalsvariabilität bei mediterranen Echiniden und Rückschlüsse auf die ökologie und Artumgrenzung fossiler Formen. Paläontol Z. 1973;47(3–4):188–216.

* View Article

* Google Scholar

85. 85. Tenuzzo BA, Zaccarelli N, Dini L. The reproductive cycle of the commercial sea urchinParacentrotus lividus(Lamark, 1816) (Echinodermata: Echinoidea) in the Ionian Sea. Italian Journal of Zoology. 2012;79(2):200–11.

* View Article

* Google Scholar

86. 86. Wangensteen OS, Turon X, Pérez-Portela R, Palacín C. Natural or naturalized? Phylogeography suggests that the abundant sea urchin Arbacia lixula is a recent colonizer of the Mediterranean. PLoS One. 2012;7(9):e45067. pmid:23028765

* View Article

* PubMed/NCBI

* Google Scholar

87. 87. Zigler KS, Lessios HA. Speciation on the coasts of the new world: phylogeography and the evolution of bindin in the sea urchin genus Lytechinus. Evolution. 2004;58(6):1225–41. pmid:15266972

* View Article

* PubMed/NCBI

* Google Scholar

Citation: Núñez-González R, Sarmiento-Lezcano AN, Caballero MJ, Tilic E, Castro-Hernández JJ (2025) Locality matters: Variation in the reproductive cycle and population structure of subtropical sea urchins. PLoS One 20(8): e0328273. https://doi.org/10.1371/journal.pone.0328273

About the Authors:

Raibel Núñez-González

Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Writing – original draft, Writing – review & editing

E-mail: [email protected]

Affiliation: Instituto Universitario EcoAqua, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain

ORICD: https://orcid.org/0000-0001-6088-3042

Airam N. Sarmiento-Lezcano

Roles: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

Affiliation: Centro Oceanográfico de A. Coruña. Instituto Español de Oceanografía, A. Coruña, Spain

ORICD: https://orcid.org/0000-0003-0567-4189

María J. Caballero

Roles: Formal analysis, Methodology, Resources, Supervision, Validation, Writing – review & editing

Affiliation: Division of Veterinary Histology and Pathology, Institute for Animal Health and Food Safety (IUSA), Veterinary School, University of Las Palmas de Gran Canaria, Arucas, Spain

Ekin Tilic

Roles: Methodology, Resources, Supervision, Validation, Writing – review & editing

Affiliation: Department of Marine Zoology, Senckenberg Research Institute and Natural History Museum, Frankfurt am Main, Germany

ORICD: https://orcid.org/0000-0003-0463-322X

José Juan Castro-Hernández

Roles: Resources, Supervision, Validation, Writing – review & editing

Affiliation: Instituto Universitario EcoAqua, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

References

1. Valentine JF, Heck KL Jr. The role of sea urchin grazing in regulating subtropical seagrass meadows: evidence from field manipulations in the northern Gulf of Mexico. Journal of Experimental Marine Biology and Ecology. 1991;154(2):215–30.

2. Ocaña A, Pérez-Ruzafa A. Los equinodermos de las costas andaluzas. Acta Granatense. 2004;3:83–136.

3. Carpenter RC. Predator and population density control of homing behavior in the Caribbean echinoid Diadema antillarum. Mar Biol. 1984;82(1):101–8.

4. Bulleri F, Benedetti-Cecchi L, Cinelli F. Grazing by the sea urchins Arbacia lixula L. and Paracentrotus lividus Lam. in the Northwest Mediterranean. Journal of Experimental Marine Biology and Ecology. 1999;241(1):81–95.

5. Pérez SA, Gil DG, Rubilar Panasiuk CT. Echinodermata. In: Calcagno JA, Editor. Los invertebrados marinos. Buenos Aires: Fundación de Historia Natural Félix de Azara Press. 2014. p. 295–316.

6. Byrne M. Annual reproductive cycles of the commercial sea urchin Paracentrotus lividus from an exposed intertidal and a sheltered subtidal habitat on the west coast of Ireland. Mar Biol. 1990;104:275--89.

7. Guillou M, Lumingas LJL. The reproductive cycle of the ‘blunt’ sea urchin. Aquaculture International. 1998;6(2):147–60.

8. Sánchez-España AI, Martínez-Pita I, García FJ. Gonadal growth and reproduction in the commercial sea urchin Paracentrotus lividus (Lamarck, 1816) (Echinodermata: Echinoidea) from southern Spain. Hydrobiologia. 2004;519(1–3):61–72.

9. Wangensteen OS, Turon X, Casso M, Palacín C. The reproductive cycle of the sea urchin Arbacia lixula in northwest Mediterranean: potential influence of temperature and photoperiod. Mar Biol. 2013;160(12):3157–68.

10. Elakkermi M, Mezali K, Soualili DL. Interpopulation variability of the reproductive cycle of Arbacia lixula (Echinodermata: Echinoidea) in the Mostaganem shallow-water area (south-western Mediterranean). Regional Studies in Marine Science. 2021;45:101810.

11. Lourenço S, José R, Neves P, Góis A, Cordeiro N, Andrade C, et al. Population Density, Reproduction Cycle and Nutritional Value of Sphaerechinus granularis (Echinodermata: Echinoidea) in an Oceanic Insular Ecosystem. Front Mar Sci. 2022;8.

12. Sala E, Zabala M. Fish predation and the structure of the sea urchin Paracentrotus lividus populations in the NW Mediterranean. Mar Ecol Prog Ser. 1996;140:71–81.

13. Vafidis D, Antoniadou C, Ioannidi V. Population Density, Size Structure, and Reproductive Cycle of the Comestible Sea Urchin Sphaerechinus granularis (Echinodermata: Echinoidea) in the Pagasitikos Gulf (Aegean Sea). Animals (Basel). 2020;10(9):1506. pmid:32858926

14. Hernández JC, Clemente S, Tuya F, Pérez-Ruzafa A, Sangil C, Moro-Abad L, et al. Echinoderms of the Canary Islands, Spain. Echinoderm Research and Diversity in Latin America. Springer Berlin Heidelberg. 2012. p. 471–510.

15. Klaoudatos D, Tziantziou L, Lolas A, Neofitou N, Vafidis D. Population Characteristics of the Upper Infralittoral Sea Urchin Arbacia lixula (Linnaeus, 1758) in Eastern Mediterranean (Central Greece): An Indicator Species for Coastal Water Quality. JMSE. 2022;10(3):395.

16. Sartoretto S, Francour P. Quantification of Bioerosion by Sphaerechinus Granularis on ‘Coralugène’ Concretions of the Western Mediterranean. J Mar Biol Ass. 1997;77(2):565–8.

17. Guidetti P, Fraschetti S, Terlizzi A, Boero F. Distribution patterns of sea urchins and barrens in shallow Mediterranean rocky reefs impacted by the illegal fishery of the rock-boring mollusc Lithophaga lithophaga. Marine Biology. 2003;143(6):1135–42.

18. Bonaviri C, Vega Fernández T, Fanelli G, Badalamenti F, Gianguzza P. Leading role of the sea urchin Arbacia lixula in maintaining the barren state in southwestern Mediterranean. Mar Biol. 2011;158(11):2505–13.

19. Tuya F, Cisneros-Aguirre J, Ortega-Borges L, Haroun RJ. Bathymetric segregation of sea urchins on reefs of the Canarian Archipelago: Role of flow-induced forces. Estuarine, Coastal and Shelf Science. 2007;73(3–4):481–8.

20. Gianguzza P, Bonaviri C. Arbacia. Developments in Aquaculture and Fisheries Science. Elsevier. 2013. p. 275–83.

21. Boudouresque CF, Verlaque M. Chapter 13 Ecology of Paracentrotus lividus. Developments in Aquaculture and Fisheries Science. Elsevier. 2007. p. 243–85.

22. Chelazzi G, Serra G, Bucciarelli G. Zonal recovery after experimental displacement in two sea urchins co-occurring in the Mediterranean. Journal of Experimental Marine Biology and Ecology. 1997;212(1):1–7.

23. Pérez-Sánchez JM, Moreno-Batet E. Invertebrados marinos de Canarias. Las Palmas de Gran Canaria: Ediciones del Cabildo Insular de Gran Canaria. 1991.

24. Alves FMA, Chícharo LM, Serrao E, Abreu AD. Algal cover and sea urchin spatial distribution at Madeira Island (NE Atlantic). Sci mar. 2001;65(4):383–92.

25. Sloan NA, Clark AM, Taylor JD. The echinoderms of Aldabra and their habitats. British Museum (Natural History). 1979.

26. Scheibling RE, Mladenov PV. The decline of the sea urchin, Tripneustes ventricosus, fishery of Barbados: a survey of fishermen and consumers. Mar Fish Rev. 1987;49(3):62–9.

27. Guidetti P, Terlizzi A, Boero F. Effects of the edible sea urchin, Paracentrotus lividus, fishery along the Apulian rocky coast (SE Italy, Mediterranean Sea). Fisheries Research. 2004;66(2–3):287–97.

28. Bertocci I, Dominguez R, Machado I, Freitas C, Domínguez Godino J, Sousa-Pinto I, et al. Multiple effects of harvesting on populations of the purple sea urchin paracentrotus lividus in north Portugal. Fisheries Research. 2014;150:60–5.

29. Lessios HA. Population dynamics of Diadema antillarum (Echinodermata: Echinoidea) following mass mortality in Panama. Mar Biol. 1988;99(4):515–26.

30. Jones GM, Hebda AJ, Scheibling RE, Miller RJ. Histopathology of the disease causing mass mortality of sea urchins (Strongylocentrotus droebachiensis) in Nova Scotia. J Invertebr Pathol. 1985;45(3):260–71. pmid:3998481

31. Dyková I, Lorenzo-Morales J, Kostka M, Valladares B, Pecková H. Neoparamoeba branchiphila infections in moribund sea urchins Diadema aff. antillarum in Tenerife, Canary Islands, Spain. Dis Aquat Organ. 2011;95(3):225–31. pmid:21932534

32. Clemente S, Lorenzo-Morales J, Mendoza J, López C, Sangil C, Alves F, et al. Sea urchin Diadema africanum mass mortality in the subtropical eastern Atlantic: role of waterborne bacteria in a warming ocean. Mar Ecol Prog Ser. 2014;506:1–14.

33. Girard D, Clemente S, Toledo‐Guedes K, Brito A, Hernández JC. A mass mortality of subtropical intertidal populations of the sea urchin Paracentrotus lividus: analysis of potential links with environmental conditions. Marine Ecology. 2011;33(3):377–85.

34. Gizzi F, Jiménez J, Schäfer S, Castro N, Costa S, Lourenço S, et al. Before and after a disease outbreak: Tracking a keystone species recovery from a mass mortality event. Mar Environ Res. 2020;156:104905. pmid:32174333

35. Hernández JC, Sangil C, Lorenzo-Morales J. Uncommon southwest swells trigger sea urchin disease outbreaks in Eastern Atlantic archipelagos. Ecol Evol. 2020;10(15):7963–70. pmid:32788953

36. Salazar-Forero CE, Reyes-Batlle M, González-Delgado S, Lorenzo-Morales J, Hernández JC. Influence of Winter Storms on the Sea Urchin Pathogen Assemblages. Front Mar Sci. 2022;9.

37. Núñez-González R, Caballero MJ, Padilla D, Martín Barrasa JL, Castro Hernández JJ. Study of histopathology on Arbacia lixula (Arbaciidae: Arbacioida) and Paracentrotus lividus (Parachinidae: Camarodonta) with bald sea urchin disease symptoms in Gran Canaria Island, Spain. Rev Biol Trop. 2024;72(S1):e58993.

38. Bertocci I, Blanco A, Franco JN, Fernández-Boo S, Arenas F. Short-term variation of abundance of the purple sea urchin, Paracentrotus lividus (Lamarck, 1816), subject to harvesting in northern Portugal. Mar Environ Res. 2018;141:247–54. pmid:30249457

39. Rey-Méndez M, Tourón N, Rodríguez-Castro B, Rama-Villar A, Fernández-Silva I, González N, et al. Crecimiento y mejora del índice gonadal en el cultivo del erizo de mar Paracentrotus lividus (Echinoida: Echinidae) en Galicia (España). Rev Biol Trop. 2015;63:261–72.

40. Tourón N, Fernández-Silva I, Rodríguez-Castro J, González NE, Catoira JL, Rey-Méndez M. Genetic Characterization ofParacentrotus lividus(Lamarck, 1816) Populations from Nw Spain and the Canary Islands. Journal of Shellfish Research. 2018;37(3):651–8.

41. Fernández-Boán M, Fernández L, Freire J. History and management strategies of the sea urchin Paracentrotus lividus fishery in Galicia (NW Spain). Ocean & Coastal Management. 2012;69:265–72.

42. Tuya F, Sanchez-Jerez P, Haroun R. Influence of fishing and functional group of algae on sea urchin control of algal communities in the eastern Atlantic. Mar Ecol Prog Ser. 2005;287:255–60.

43. Clemente S, Hernández JC, Brito A. Evidence of the top–down role of predators in structuring sublittoral rocky-reef communities in a Marine Protected Area and nearby areas of the Canary Islands. ICES Journal of Marine Science. 2008;66(1):64–71.

44. QGIS Development Team. QGIS Geographic Information System (Version 3.14 “Pi”). Open Source Geospatial Foundation Project. 2020. Available from: http://www.qgis.org

45. GEBCO Compilation Group. GEBCO 2024 Grid. 2024. Available from: https://doi.org/10.5285/38a552b8-2c35-472f-e053-6c86abc06b44

46. Cooper JE. Anesthesia, analgesia, and euthanasia of invertebrates. ILAR J. 2011;52(2):196–204. pmid:21709312

47. Wahltinez SJ, Stacy NI, Hadfield CA, Harms CA, Lewbart GA, Newton AL, et al. Perspective: Opportunities for advancing aquatic invertebrate welfare. Front Vet Sci. 2022;9:973376. pmid:36458054

48. Bancroft JD, Layton C, Suvarna SK. Bancroft’s Theory and Practice of Histological Techniques. New York: Churchill Livingstone. Elsevier. 2013.

49. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3(5):294–9. pmid:7881515

50. Duran S, Palacín C, Becerro MA, Turon X, Giribet G. Genetic diversity and population structure of the commercially harvested sea urchin Paracentrotus lividus (Echinodermata, Echinoidea). Mol Ecol. 2004;13(11):3317–28. pmid:15487992

51. Geneious v.11.0.2. Biomatters. 2023. Available from: https://www.geneious.com

52. Legendre P. Model II regression user’s guide, R edition. 14. 1998.

53. Warton DI, Duursma RA, Falster DS, Taskinen S. smatr 3 - an R package for estimation and inference about allometric lines. Methods in Ecology and Evolution. 2012;3:257–9.

54. Selden R, Johnson AS, Ellers O. Waterborne cues from crabs induce thicker skeletons, smaller gonads and size-specific changes in growth rate in sea urchins. Mar Biol. 2009;156(5):1057–71. pmid:24489404

55. Lenth R. Emmeans: Estimated Marginal Means, aka Least-Squares Means. 2025.

56. Sachs L. Applied Statistics: A Handbook of Techniques. New York: Springer-Verlag. 1982.

57. Sokal R, Rohlf F. Biometry, the principles and practice of statistics in biological research. 4th ed. New York: Freeman and Co. 2012.

58. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. 2023.

59. Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 2017;18(1):529. pmid:29187165

60. Spirlet C, Grosjean P, Jangoux M. Reproductive cycle of the echinoidParacentrotus lividus: analysis by means of the maturity index. Invertebrate Reproduction & Development. 1998;34(1):69–81.

61. Wickham H, François R, Henry L, Müller K, Vaughan D. dplyr: A Grammar of Data Manipulation.

62. Wickham H, Vaughan D, Girlich M. tidyr: Tidy Messy Data. 2024.

63. Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. 2016.

64. Felden J, Möller L, Schindler U, Huber R, Schumacher S, Koppe R, et al. PANGAEA - Data Publisher for Earth & Environmental Science. Sci Data. 2023;10(1):347. pmid:37268655

65. Núñez-González R, Sarmiento-Lezcano AN, Tilic E, Caballero Cansino MJ, Castro Hernández JJ. Biometric measurements of dominant subtropical sea urchins in the northeast Atlantic Ocean (June 2020–May 2021). PANGAEA. 2025.

66. Núñez-González R, Sarmiento-Lezcano AN, Tilic E, Caballero Cansino MJ, Castro Hernández JJ. Oocyte dimensions of dominant subtropical sea urchins in the Northeast Atlantic Ocean (June 2020–May 2021). PANGAEA. 2025.

67. Walker CW, Unuma T, McGinn NA, Harrington LM, Lesser MP. Reproduction of sea urchins. Developments in Aquaculture and Fisheries Science. Elsevier. 2001. p. 5–26.

68. Walker CW, Lesser MP, Unuma T. Gametogenesis in regular sea urchins: Structural, functional, and molecular/genomic biology. Developments in Aquaculture and Fisheries Science. Elsevier. 2020. p. 29–50.

69. Khaili A. Reproductive cycle of the sea urchin Paracentrotus lividus (Lamarck, 1816) from the Moroccan western Mediterranean Sea: histology, gonadal index and size at first sexual maturity. Egypt J of Aquatic Biolo and Fish. 2023;27(2):745–71.

70. Herrero Barrencua A. Aspectos reproductivos del erizo común (Paracentrotus lividus) en aguas del Este de Gran Canaria. Universidad de Las Palmas de Gran Canaria. 2008. https://accedacris.ulpgc.es/bitstream/10553/11652/2/0697866_00000_0000.pdf

71. Privitera D, Chiantore M, Mangialajo L, Glavic N, Kozul W, Cattaneo-Vietti R. Inter- and intra-specific competition between Paracentrotus lividus and Arbacia lixula in resource-limited barren areas. Journal of Sea Research. 2008;60(3):184–92.

72. Verlaque M. Preliminary data on some Posidonia feeders. Rapp PV Réun Commiss Internatl Explor Sci Méditerr. 1981;27(2):201–2.

73. González-Irusta JM, Goñi de Cerio F, Canteras JC. Reproductive cycle of the sea urchinParacentrotus lividusin the Cantabrian Sea (northern Spain): environmental effects. J Mar Biol Ass. 2009;90(4):699–709.

74. Lozano J, Galera J, López S, Turon X, Palacin C. Biological cycles and recruitment of Paracentrotus lividus (Echinodermata: Echinoidea) in two contrasting habitats. Mar Ecol Prog Ser. 1995;122:179–91.

75. Loi B, Guala I, Pires da Silva R, Brundu G, Baroli M, Farina S. Hard time to be parents? Sea urchin fishery shifts potential reproductive contribution of population onto the shoulders of the young adults. PeerJ. 2017;5:e3067. pmid:28289567

76. González-Henríquez N, Rey-Méndez M, Catoira Gómez JL, Ruiz de la Rosa M, Louzara Fernández G, Girard Berard D. Evolución temporal del índice gonadal de los erizos marinos Paracentrotus lividus (Echinoida: Echinidae) y Diadema africanum (Diadematoida: Diadematidae) en las islas Canarias (España). Rev Biol Trop. 2015;63:251–60. http://dx.doi.og/10.15517/rbt.v63i2.23160

77. Girard Berard D. Dinámica poblacional del erizo de mar Paracentrotus lividus (Lamarck, 1816) en las islas Canarias: estudio de la biología, ecología y valoración del estado de las poblaciones. Universidad de La Laguna. 2022.

78. Soualili DL, Guillou M, Semroud R. Age and growth of the echinoid Sphaerechinus granularis from the Algerian coast. J Mar Biol Ass. 1999;79(6):1139–40.

79. Guillou M, Michel C. The influence of environmental factors on the growth of Sphaerechinus granularis (Lamarck) (Echinodermata: Echinoidea). Journal of Experimental Marine Biology and Ecology. 1994;178(1):97–111.

80. Kelly MS, Owen PV, Pantazis P. The commercial potential of the common sea urchin Echinus esculentus from the west coast of Scotland. Hydrobiologia. 2001;465(1–3):85–94.

81. Starr M, Himmelman JH, Therriault J-C. Environmental Control of Green Sea Urchin,Strongylocentrotus droebachiensis, Spawning in the St. Lawrence Estuary. Can J Fish Aquat Sci. 1993;50(5):894–901.

82. Reyes-Luján J, Zapata-Vívenes É, Arrieche D, Lodeiros C. Efecto de la estacionalidad sobre el desarrollo gonadal y la composición bioquímica del erizo de mar Echinometra lucunter lucunter (Echinodermata: Echinometridae) en el Islote Lobos, Venezuela. Gayana. 2024;88(1):44–56.

83. Arístegui J, Tett P, Hernández-Guerra A, Basterretxea G, Montero MF, Wild K, et al. The influence of island-generated eddies on chlorophyll distribution: a study of mesoscale variation around Gran Canaria. Deep Sea Research Part I: Oceanographic Research Papers. 1997;44(1):71–96.

84. Ernst G, Hähnel von W, Seibertz E. Aktuopaläontologie und Merkmalsvariabilität bei mediterranen Echiniden und Rückschlüsse auf die ökologie und Artumgrenzung fossiler Formen. Paläontol Z. 1973;47(3–4):188–216.

85. Tenuzzo BA, Zaccarelli N, Dini L. The reproductive cycle of the commercial sea urchinParacentrotus lividus(Lamark, 1816) (Echinodermata: Echinoidea) in the Ionian Sea. Italian Journal of Zoology. 2012;79(2):200–11.

86. Wangensteen OS, Turon X, Pérez-Portela R, Palacín C. Natural or naturalized? Phylogeography suggests that the abundant sea urchin Arbacia lixula is a recent colonizer of the Mediterranean. PLoS One. 2012;7(9):e45067. pmid:23028765

87. Zigler KS, Lessios HA. Speciation on the coasts of the new world: phylogeography and the evolution of bindin in the sea urchin genus Lytechinus. Evolution. 2004;58(6):1225–41. pmid:15266972