Salinity patterns and local migration determine the isotopic composition of the invasive blue crab, Callinectes sapidus, along the Spanish Mediterranean coast

Abstract

δ¹³C and δ¹⁵N patterns of the blue crab, Callinectes sapidus, and its potential diets, were investigated in eleven localities within four invaded regions of the Spanish Mediterranean (Catalonia, Valencia, Alicante, and Murcia) subjected to contrasting salinities and degrees of connectivity with the open sea. These regions host blue crab fisheries of variable importance that might be regulated by availability of food resources and local environmental conditions. When present, large adults and immature, subadult sizes of each sex were captured to evaluate possible differences associated to age movement across habitats. SIBER and MixSIAR were used to assess patterns of isotopic niche and dietary contributions. Results showed significant effects for all factors and interactions (except for Sex and Sex x Size in δ¹⁵N). The effect size in Region (η_p² = 0.82 and 0.70, respectively for δ¹³C and δ¹⁵N) and Locality (η_p² = 0.53 and 0.46), was substantially greater than that of crab Size (η_p² = 0.37 and 0.21) and Sex (η_p² = 0.06 and 0.02), concurring with increasing levels of isotopic niche overlap (9% and 11% in Locality and Region, 32% in Size and 44% in Sex). MixSiar results indicated strongly variable contributions from food sources at each locality (TPs from 2.3 to 3.6), but with higher quantity of fish, algae, and crustaceans (27.7%, 18% and 15.1%). Dietary results showed little relation with isotopic patterns, whereas significant associations were found between local salinities and signatures in both crabs (R² = 0.518 and 0.757, for δ¹³C and δ¹⁵N) and diets. Overall, our study suggests that blue crab habitat use in small Mediterranean estuaries might largely differ from native areas, with movements being mostly restricted to young individuals and/ or certain localities with higher connectivity with the open sea (e.g., the Ebro River). Salinity conditions emerge as a major variable shaping isotopic patterns of populations on a large scale.

Introduction

The blue crab (Callinectes sapidus Rathbun) is considered one of the 100 most dangerous invasive species in the Mediterranean Sea, capable of altering the biodiversity and trophic functioning of native communities, and causing economic and social losses to local fisheries, aquaculture, and traditional practices [1–3]. In Spain, the national legislation has not included the species in the Spanish Catalog of Invasive Exotic Species as the only means to market the blue crab and control its spread [4]. Originally native to the western Atlantic Ocean, from Nova Scotia to Argentina [5], the blue crab was introduced to the Eastern Mediterranean Sea in 1948 [6], likely through ballast water discharge from ships or via the Suez Canal. Mancinelli et al. [7] highlighted the ubiquitous spread of the species, with up to 458 records in the Mediterranean Sea, particularly in Spain, Italy, and Greece. Researchers emphasize that an anomalous accumulation of records has occurred in Spain during the last 10 years, given that the first population of the species in the Tancada lagoon (Ebro Delta, Catalonia) was found in 2012 [8], which is comparatively much more recent than those in Greece and Italy dating from the late 40s. In fact, the blue crab has experienced a very quick expansion along the Spanish Mediterranean coast. A single specimen was first detected in 2004 in Mar Menor (Murcia), followed by the Ebro Delta population in 2012, and since then, it has reached the Albufera lagoon (Valencia) in 2013, Gandía also in Valencia in 2014, the Segura River (Alicante) in 2015 [9, 10] and the Balearic Islands in 2017 [11]. In late 2018, the blue crab was found on the Southern Atlantic coast of Portugal [12], despite the absence of estuarine habitats between the Mar Menor and the Gibraltar strait. In fact, recent research by González-Ortegón et al. [13], indicate that only two different haplotypes are present along the Spanish Mediterranean coast, one haplotype being dominant from the Ebro Delta to the Mar Menor, and a second haplotype being more representative of Alboran Sea populations, featuring high connectivity with the Gulf of Cadiz.

The blue crab is regarded as a generalist omnivore, feeding on any available food resource depending on local availability [14]. Hines [15], conducted an extensive review of stomach contents and other feeding observations and indicated that the blue crab diet might include at least 99 species from several phyla, especially mollusks (20–40%), arthropods (10–26%), chordates (fishes; 5–12%) and annelids (polychaetes; 1–7%). Additionally, the diet often includes significant contributions of plant material (1–20%) and algae (3–30%), as well as sediments (up to >50%) when prey items become scarce [16]. Yet, the blue crab might also change during development, with juveniles inhabiting shallower waters feeding on a variety of small epibiota and infauna, and large adults targeting larger and less diverse prey [17, 18]. Miller et al. [19] also indicated that the consumption of bivalves was the highest (39%) for subadult (60–119 mm) and adult sizes (≥120 mm), whereas recruits (≤59 mm) ingested significantly higher proportions of plant matter (10–12%). Sex might be another source of dietary variability, since microhabitat partitioning is often reported in estuarine mating environments, with large males being captured in shallower or lower salinity areas than mature females [20, 21]. Also, the species features sexual differences in claw morphology which further suggest possible variability in dietary habits [22]. After copulation, female blue crabs are renowned for their spawning migration from the upper estuary to higher salinity waters by the mouths of estuaries and coastal areas [23], which involves further movement across habitats with distinctive food resources. Female blue crabs do not often survive after larval release at high salinities [24], whereas males in estuarine waters may continue to molt and grow for 1–3 additional instars (typical large size is 180–200 mm, but some might grow up to >250 mm; [15]). Larval development includes seven zoea instars, followed by a single megalopal stage that return to settle and metamorphose in submerged macrophytes in the lower estuary, and from there juveniles start dispersing upstream, to lower salinity habitats [25].

Stable isotope analyses (SIA) offer a powerful tool to simultaneously assess the dietary behavior of consumers and their migration patterns. The isotopic niche, as analyzed through the Stable Isotope Bayesian Ellipses (SIBER) framework in R, provides a method to compare the isotopic niches of different populations and quantify their dietary overlap based on their stable isotope compositions [26, 27]. Furthermore, δ¹⁵N and δ¹³C signatures and variability in both isotope data can be used to feed mixing models such as MixSIAR to estimate the proportions of different food sources in an organism’s diet and the associated uncertainty [28, 29]. δ¹⁵N signatures can also offer an accurate estimate of the trophic position of a species within the hierarchy of local food webs given that a baseline indicator is provided [3, 30]. Additionally, because freshwater signatures of primary producers are typically more depleted in δ¹³C than those in estuarine and marine environments (by 5 to >9‰) [31, 32], isotopic patterns are effectively transmitted through the entire food web across salinity gradients and can provide useful evidence of habitat use [32–36]. In blue crab, SIA has provided valuable information on the origin of δ¹³C from dietary sources across different habitats through ontogenic stages [37]. In invaded areas, it has helped to understand the functional traits and potential impacts of the species on native fauna through estimation of the trophic position and its isotopic niche [3, 38].

In this context, the objective of the present study was twofold. First, we aimed to assess the dietary habits of the blue crab in invaded Spanish Mediterranean regions, including Catalonia, Valencia, Alicante and Murcia. Within each region, we selected different localities featuring contrasting habitats (lagoons, bays, estuaries, open sea, and agricultural drainage channels), with variable salinity and connectivity conditions, in terms of distance from the open sea and/ or presence of regulated gates. Given that most processes affecting the different life-stages of invertebrates are scale-dependent, evaluation at two different spatial scales (ca. 10s vs. 100s of km) might help to understand functional patterns. Second, we aimed to evaluate differences in diet associated to individual age (immature subadults vs. large adult sizes) and sex-related habits. δ¹⁵N and δ¹³C signatures of blue crabs were used to assess variability associated to the spatial scale and demographic factors, to build the size and magnitude of overlap of the isotopic niches, and to determine the relative influence of salinity in overall patterns. Furthermore, the dietary composition (mixing models) and trophic position (when possible) were assessed in order to validate observed spatial patterns in stable isotopes. Variability in the isotopic signatures between sizes and sexes that could be associated to short-term mobility patterns was also assessed for each locality, taking into account local connectivity features.

Materials and methods

Study sites

The study was conducted in four regions along the mainland of the Spanish Mediterranean coast: Catalonia, Valencia, Alicante, and Murcia (North to South). Within each region, three localities (only 2 in Murcia) where the blue crab is commonly observed and/or where there are well-established fishery practices targeting the species were selected (i.e., a total of 11 localities). In Catalonia, these localities included (1) an olygohaline to mesohaline stretch of the lower Ebro River between St. Jaume d’Enveja and the Riumar Port; and (2) polyhaline waters of the Encanyissada coastal lagoon, connected to the (3) Alfacs Bay through the Saint Pere channel (Fig 1A) and receiving freshwater discharges from adjacent rice fields that comprise up to 70% of the Ebro Delta surface. In Valencia, localities were selected in the Natural Park of L’Albufera, an area that includes a shallow coastal lagoon and surrounding wetlands connected to the Mediterranean Sea through three one-way gated channels that allow the maintenance of freshwater levels [25]. (4) The Pujol area is within the oligohaline lagoon, whereas (5) the Perelló channel (polyhaline) drains the waters from the Albufera to the open Mediterranean Sea (6) (Fig 1B). In Alicante, localities included (7) Cola del Río at the mouth of the Segura River, and the (8–9) Convenio and Orones channels in the Natural Park of El Hondo located about 10 km inland (Fig 1C), the three of them slightly above the upper limit of the oligohaline stretch. The Natural Park of El Hondo is a wetland system composed of different lagoons and channels, which receives most of its water from the Segura River and aquifer waters, and is surrounded by agricultural lands that have caused severe eutrophication and the disappearance of most of the submerged vegetation following intensive aquifer exploitation and water quality degradation [39, 40]. Finally, in Murcia, two localities were selected in the hypersaline waters of the Mar Menor, the largest coastal lagoon of the Mediterranean basin (Fig 1D). These localities were (10) Seco Grande and (11) Encañizada, both placed in the seaward side of the Mar Menor, further away from areas of agricultural freshwater discharges causing the eutrophication of the lagoon [41]. The Seco Grande is a sand shoal located north of the only navigable channel between the lagoon and the Mediterranean, whereas the Encañizada is a very shallow area with complex water exchange through numerous channels distributed within a lattice of sandbanks and islets.

[Figure omitted. See PDF.]

a) Ebro Delta localities (Catalonia); b) Albufera localities (Valencia); c) Segura River and Hondo Lagoon localities (Alicante); and d) Mar Menor lagoon localities (Murcia).

Blue crab and food sources sampling

Ethics statement. Research conducted in the present study was not experimental. The capture or purchase of commercial fish, blue crabs and other invertebrates in Spain do not require ethics approval and the involvement of an animal research ethics committee. Animals were kept for a couple of hours at -20°C before dissection of muscle tissue.

Blue crabs were captured between mid-June to late August 2022 by deploying either crab traps of different models or gillnets, depending on local fishing gears used across the study regions. At the Riumar locality in the Ebro Delta (Catalonia), where a localized fishery operates in the selected oligohaline to mesohaline stretch of the Ebro River (St. Jaume d’Enveja to Riumar), crabs were obtained directly from the fishermen. The traps were deployed in the morning and collected 24 h later. This operation was repeated as needed to obtain different sexes and sizes. After collection, crabs were put into nylon nets with plastic labels and transported back to the lab in an ice cooler.

At each location we aimed to capture individuals of both sexes within two different size ranges: large adults (120 to 180 mm carapace width with spines), and immature, subadult individuals (50 to 100 mm carapace width, also with spines). However, this was not always possible due to differences in ontogenic and seasonal distribution of crabs, which resulted in certain absences during the study period (see Table 1 for details on the number of individuals of each sex and size obtained at each location). Samples of muscle tissue were consistently extracted from one of the claws and dried at 60 ⁰C until reaching a constant weight. Then they were reduced to powder with a mortar and pestle. Grinding tools were carefully rinsed with ethanol 99% after each sample to remove any remains before reuse. It should be noted that our results are temporally constricted to an estimate isotopic turn-over in blue crab tissues of ca. 3 months [42] during the summer season.

[Figure omitted. See PDF.]

Salinity was measured in all localities at the moment of deployment using a multiparametric probe equipped with a data logger, although brands differed among regions. When available (Riumar and Alfacs in Catalonia, and Pujol and Perelló in Valencia), more integrative temporal data across the entire summer period from other ongoing monitoring was also used. When samples were obtained from commercial catches, salinity was measured across the entire fishing ground to obtain spatial averages.

Benthic species of fish and small crustaceans were captured in June-July by deploying three two-meter long fyke nets with 80 cm hoop diameter and 5 mm mesh size were set in shallow areas (60–100 cm depth) in the different localities. Fyke nets were set in late afternoon and hauled the next morning (12 h soaking time) and captures transported to the lab in an icebox for further identification and sorting. Fish and crustaceans were dissected to obtain the musculature and then samples rinsed with ultrapure water to prevent contamination with other tissues.

Bivalve samples of mussel (Mytilus galloprovincialis) and oyster (Crassostrea gigas) in the Alfacs Bay (Catalonia) were obtained from the local producers. In all localities, infauna (bivalves and Polychaeta) and benthic gastropods were sampled with a hand rake coupled to a net of 0.5 cm mesh. When available, the anemone Paranemonia cinerea growing on leaves of Cymodocea nodosa was collected by scraping them off with a razor blade. Once in the laboratory, gastropods and bivalves were removed from their shells and all samples rinsed with ultrapure water to remove sediments.

The most abundant species of plant and algae in each locality were either collected by hand or with a Van Veen grab and identified and sorted to species level under the stereomicroscope. The top 2 cm of the sediment layer including an inorganic fraction and detrital material with bacterial/microalgae biofilms was collected (N = 3 per site) using plastic containers. In the lab, samples were homogenized and split in two for the removal of inorganic carbon following the ISO 10694 normative [16].

All animal and vegetal food sources were classified into different groups depending on local availability: fish, crustaceans (crabs and/or prawns), bivalves, gastropods, anemones, odonata larvae, polychaetes, plants, and algae. For each taxon, a minimum of three individuals or vegetation patches was required for inclusion in a given group and considered as an indicator of medium to high availability at the study locality (see S1 Dataset for details on taxa composition). Diet items were dried at 60⁰C, reduced to powder, and weighed. For each locality, an equal quantity of a given taxon (N = 3 for each one collected) was mixed together with other taxa belonging to the same trophic group, to obtain a total of N = 3 replicates (see [16] for a similar approach). Some trophic groups such as anemones and odonata larvae were composed of just one species.

Stable isotope analyses

Samples were analyzed by isotopic ratio mass spectrometry (IRMS) at the research support service (SAI) of the University of A Coruña, using an elemental analyzer FlashEA1112 coupled with an Interface Conflo II (ThermoFinnigan) to an isotopic ratio mass spectrometer Deltaplus (ThermoFinnigan). Isotope ratios in samples were calculated from linear calibration curves constructed with standard reference materials of known composition and a blank correction. The difference in isotopic composition between the sample and reference material is determined by the equation:where R _sample is the ¹³C/¹²C or ¹⁵N/¹⁴N in the sample; R _standard is the ¹³C/¹²C or ¹⁵N/¹⁴N in the calibration material and δ _{sample-standard} is the difference in isotopic composition of the sample relative to that of the reference (Vienna Pee Dee Belemnite for carbon, and atmospheric nitrogen for nitrogen). Experimental precision based on the standard deviation of replicates of acetanilid standard was considered to be adequately high (±0.15 ‰; n = 10).

δ¹³C signatures were normalized for the effect of lipid storage using the equation with C:N ratios indicated by [43] for aquatic animals (corrected δ¹³C = δ ¹³C - 3.32 + 0.99 x C:N), and for vegetal samples (corrected δ¹³C = δ ¹³C + 1.25–0.00 x C:N). Corrections were applied whenever δ¹³C values increased by at least 0.1‰ [44].

Isotopic data analyses

Differences in the composition of normalized δ¹³C and δ¹⁵N signatures of blue crabs across Regions (Fixed factor, 4 levels), Localities (Fixed factor, 11 levels, nested in Region), Sex (Fixed factor, 2 levels, nested in Location), and Size (Fixed factor, 2 levels, nested in Location) were investigated with a 4-way mixed ANOVA and SNK post-hoc analysis. The significance of nested factors was assessed through estimated marginal means results. Further effects of Sex and Size at each locality were also assessed with a two-way factorial ANOVA (4 for δ¹³C and 4 for δ¹⁵N) or one-way ANOVA (5 for δ¹³C and 5 for δ¹⁵N), depending on samples’ availability.

For all ANOVAs, normality (Chi-square test) and homogeneity of variances (Cochran’s test) were tested. All ANOVA were performed using the GLM package in R. The possible relationships between normalized δ¹³C and δ¹⁵N in blue crabs and diet groups (N≥ 4 localities) and local salinities were assessed using Pearson’s correlation analysis.

Blue crab isotopic niche

The package SIBER (Stable Isotope Bayesian Ellipses in R) [26] was used to assess food web structure. Standard ellipses corrected for small sample size (SEAc) were used to represent blue crab samples in the isotopic space. SEAc in units of area (‰²) features the same properties of the Total Area (TA) of the convex polygon but it is unbiased with respect to sample size [26]. Then, the Bayesian estimate of the standard ellipse and its area (SEAb) was calculated for each factor (Region, Locality, Sex, and Size) using Markov-Chain Monte Carlo (MCMC) simulations to provide a measure of the uncertainty associated with the standard ellipse area estimate. This method samples randomly replicated sequences in the 95% confidence interval for the value distribution for both stable isotopes (δ¹³C and δ¹⁵N) to correct the bivariate ellipses. The magnitude of isotopic overlap (‰²) across factors levels was obtained from SEAb estimations and expressed as a % of the total area of two given factor levels overlapping each other.

Estimates of trophic position

The trophic position (TP hereafter) of blue crab was estimated when possible, according to the methodology and equation proposed by [3, 45]:Where δ¹⁵N Consumer is the nitrogen isotopic signature of the blue crab, δ¹⁵N baseline is the mean isotopic signature of locally available bivalves as well-known primary consumers (primary producers = trophic level 1, primary consumers = trophic level 2, and so on). Bivalves, and particularly the Mediterranean mussel, have been commonly used as food web baseline in previous stable isotope studies [3, 16, 46–48] and therefore allow for a comparative approach. Δ¹⁵N is the δ¹⁵N isotopic enrichment for the blue crab, and λ the trophic level of the baseline indicator, respectively. TP could only be estimated in 4 of the 11 localities (Encanyissada lagoon, Alfacs, Open sea, and Encañizada), were bivalves could be found.

Isotope mixing models

The contribution of local resources to blue crab diets was assessed with the MixSIAR Bayesian Mixing Models (V.3.1) R package [29], using a generalist prior distribution. Models were conducted separately for each locality to avoid possible confounding effects due to large variability in salinity that could strongly influence δ¹³C signatures. For food resources we used the proxy value obtained for each locally available trophic group. For fractionation we applied the 0.4‰ Δ¹³C and the 3.4‰ Δ¹⁵N values given for non-herbivorous aquatic consumers [30], as they have been largely used for blue crab in previous work [3, 16, 45, 49]. Runs were also conducted using the 1.3‰ SD of δ¹³C and 1‰ δ¹⁵N indicated by Post [30].

Prior to the application of mixing models, an isospace plot was made to verify that consumer δ¹⁵N and δ¹³C values fell within the prey polygon in isospace [28]. The Markov Chain Monte Carlo (MCMM) in MixSiar was set as follows: chain length: 100,000; burn-in: 50,000; thin: 50; and number of chains: 3. With these settings, convergence conditions for the Gelman–Rubin diagnostic was <1.05 in all cases; and in the Geweke diagnostic testing for the equality of the means in the first and last part of the Markov chains, the number of variables falling outside the ±1.96 range was < 5% for the three chains. Results corresponding to the 50% quartile were considered as the median source contribution for each diet source along with SD for comparative purposes.

In order to visually assess the similarity between isotopic and dietary patterns at each locality, MixSiar results for median source contributions were squared root transformed and plotted in a nMDS ordination based on Euclidian distances using the PRIMER-6 software package (Primer-E Ltd, Plymouth, UK).

Results

Isotopic signatures of blue crab across spatial scales, sex and sizes

Patterns of δ¹³C showed significant variability across Regions, Localities, Sex, Size and the Sex- Size interaction, although spatial effects were the most important (Fig 2; Table 2). The highest values were recorded in Murcia (Mur: -13.58 ± 0.25‰), followed by Catalonia (Cat: -20.34 ± 0.13‰), Valencia (Val: -24.28 ± 0.18 ‰) and Alicante (Ali: -25.49 ± 0.37‰). Across Localities, results of estimated marginal means indicated equally high values in Encañizada and Seco Grande (Mur); followed by Alfacs (Cat); the Encanyissada lagoon (Cat); Pujol (Val), Riumar (Cat), and Cola del Río (Ali); the Open Sea (Val); Perelló (Val); Convenio (Ali) and lowest in Orones (Ali). For Sex, males displayed higher values than females (-20.71 ± 0.16‰ vs. -22.28 ± 0.17‰, respectively), and for Size, small immature sizes higher than large adults (-19.56 ± 0.20‰ vs. -23.36 ± 0.12‰, respectively). In particular, the δ¹³C signature of small males was slightly higher than that of small immature females (-17.76 ± 0.27‰ vs. -20.97 ± 0.28‰), and a similar pattern was observed between large adult males and females (-23.01 ± 0.18‰ vs. -23.77 ± 0.17‰).

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Error bars are SE.

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For δ¹⁵N, results of the 4-way ANOVA were significant for Region, Locality and Size (Fig 2; Table 2). At the regional level, the highest values were observed in Alicante (Ali: 16.40 ± 0.29‰), followed by Valencia (Val: 13.58 ± 0.14‰), Catalonia (Cat: 12.69 ± 0.10‰), and the lowest in Murcia (Mur: 8.03 ± 0.20‰). At local levels, the highest values were detected in Cola del Río (Ali); Orones and Convenio (Ali); Pujol (Val) and Riumar (Cat); Open Sea (Val); Perelló (Val) and Encanyissada lagoon (Cat); Alfacs (Cat); Seco Grande (Mur), and the lowest in the Encañizada (Mur). For Size, a significant enrichment in δ¹⁵N was observed from small immature individuals (12.43 ± 0.16‰) to large adults (13.73 ± 0.10‰).

2-way ANOVA results of δ¹³C signatures at each locality (Table 3) helped to identify where Size and Sex effects patterns were stronger. In four localities (Alfacs, Encanyissada, Riumar (all three Cat) and Pujol (Val)), small individuals featured higher δ¹³C signatures than large ones, whereas no effects were observed in Perelló (Val), Cola del Río (Ali), Encañizada and Seco Grande (both Mur) and two localities only featured large females (Open sea, Val) or large males: Orones (Ali). Only one locality (Convenio, Ali) featured Sex effects, and a Size x Sex interaction was also observed in the Encanyissada lagoon and in Riumar featuring higher values in small males, and lowest in large males (Table 3).

[Figure omitted. See PDF.]

Sex (F = female; M = male), Size (S = small; L = large). One-way ANOVAs with locally available sizes and sexes are indicated with superscripts 1–4. The two localities where only large females (Valencia, open sea) or large males (Alicante, Orones) were available are not indicated. Significant p-values are indicated in bold.

¹ Small males absent, ² Small individuals absent, ³ Large females absent, ⁴ Large individuals absent.

For δ¹⁵N, significant Size or Sex effects were only observed in four localities: Alfacs and Riumar (both Cat), Perelló (Ali), and Seco Grande (Mur) (Table 3). Despite an overall effect of Size (Table 2), with significantly higher values in large individuals, this trend was only observed in Perelló (Val) and Seco Grande (Mur), whereas Alfacs and Riumar (Cat) featured opposite effects, with significantly higher values in immature sizes.

A significant association between isotopic signatures of blue crab and salinity was found for both δ¹³C (df = 10, F = 9.69, R² = 0.518; p = 0.012) and δ¹⁵N (df = 10, F = 28.03, R² = 0.756; p = 0.0005) (Fig 3), consistent with the spatial aggregation of samples observed in Fig 2. Similar but more inconsistent patterns across isotopic signatures were found for some diet groups (pooled taxa), including sediments (δ¹⁵N: df = 10, F = 50.61, R² = 0.849; p = 0.00005), plants (δ¹³C: df = 7, F = 10.64, R² = 0.63; p = 0.0173; δ¹⁵N: F = 17.65, R² = 0.74; p = 0.0056), fish (δ¹⁵N: df = 10, F = 9.39, R² = 0.510; p = 0.0134), and bivalves (δ¹⁵N: df = 3, F = 22.50, R² = 0.918; p = 0.0416), but not in algae (df = 6), crustaceans (df = 7), and polychaeta (df = 3).

[Figure omitted. See PDF.]

See text for details on R and p values.

Isotopic niches of blue crab

Bivariate standard ellipses representative of the isotopic niche of blue crab (size-corrected SEAc) also indicated significant variability in distribution and size across Regions, Localities, Sex, and Sizes (Fig 4; Table 4). Among Regions, Catalonia featured the largest size, followed by Alicante and Valencia, and the lowest in Murcia (36.4%, 54.1%, and 63.9% smaller, respectively), although maximum and minimum sizes were attained in Cola del Río (Ali), and the Open Sea (Val)(Fig 5A, 5B). For Sex and Size, males featured larger isotopic niche than females (by 20.7%), and small immature individuals larger than large adults (by 27.8%) (Table 4, Fig 5C, 5D).

[Figure omitted. See PDF.]

Isotopic niche of C. sapidus for the different study factors: A) Regions; B) Locality; C) Sex; and D) Size. Points: individual δ¹³C and δ¹⁵N measurements, dashed lines: total area of the convex hull, colored solid lines: standard ellipse areas corrected for small sample size (SEAc).

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Boxplots of the SIBER Bayesian ellipse areas (SEAb in ‰²) of blue crab for the different study factors: A) Region; B) Locality; C) Sex; and D) Size. 50, 75 and 95% credibility intervals are indicated, with dots representing the modes of the Bayesian distributions. The cross indicates the standard ellipse areas calculated using the algorithm for small sample sizes (SEAc).

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TA: total area of the convex polygon; SEA: the standard ellipse area; SEAc: standard ellipse area corrected for small sample size.

The significance of the ellipses overlap varied notably among study factors (see S2 Dataset). For Region there was an average overlap of 11.08% considering the total area of any combination of two ellipses, with maximum rates between Cataluña and Valencia (26.47%), and between Valencia and Alicante (22.43%) and no overlap between Murcia and Alicante and Valencia (Fig 4A). For Locality the average overlap was also low (9.35%), but it ranged from 31.47% (between Encañizada and Seco Grande in Murcia) to 31.03% (between Pujol (Val) and Riumar (Cat)), to no overlap between Riumar (Cat), Valencia, and Alicante localities, and the two localities in Mar Menor (Mur) (Fig 4B). The magnitude of overlap was the greatest for Size (32% between subadult and adult sizes), and Sex (44% between males and females) (Fig 4C, 4D), consistent with the size of partial eta squared values in the 4-way ANOVA.

Trophic position and MixSiar model

The trophic position (TP) of blue crab could only be estimated in 4 of the study localities (Alfacs, and the Encanyissada lagoon in Catalonia, Open Sea in Valencia, and Seco Grande in Murcia) where bivalves could be found and used as a baseline for the calculation. In the Alfacs Bay, the TP of small individuals was 1.26, whereas in Seco Grande the opposite pattern was observed with large individuals featuring 1.21 times higher TP than small individuals, and no clear differences were observed in the Encanyissada lagoon (Table 5). Overall, the highest TP was detected in the Open Sea, whereas in the other localities values varied from 2.4 in the Encanyissada lagoon, 2.58 in the Alfacs Bay (both Cat), and 2.68 in Seco Grande (Mur). These values are slightly lower than those previously recorded in Catalonia using the same methods, particularly for the open sea (Table 5).

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Data are only shown for those sites in which bivalves were available. For the Catalonian region, TP values previously found in the Alfacs Bay and other habitats of the Ebro Delta are also shown [16, 49] for further comparison.

MixSiar model results showed that fish, algae, and crustaceans were the primary food items across locations (Table 6), with average contributions of 25.7%, 18%, and 15.1%, respectively. Other items with contributions between 5 to 7% were plants, sediments, Polychaeta, and bivalves, whereas Actiniaria and Odonate larvae were constricted to certain locations and could be considered occasional items (Table 6). Importantly, however, similarities in dietary composition across localities plotted in nMDS (Fig 6) did not appear to mirror those previously described for isotopic patterns (e.g. the two localities in Murcia are positioned far apart in the nMDS but feature very similar isotopic composition, and a similar incongruence is observed for Open Sea and Pujol (both Val), among others; see Figs 2 and 6).

[Figure omitted. See PDF.]

[Figure omitted. See PDF.]

We used the Δ¹⁵δN of 3.4 and a Δ¹³δC of 0.4 given for non-herbivorous aquatic consumers [30], and previously used in blue crab [3, 16, 45, 49].

Discussion

In spite of the large spatial scale evaluated along Spanish Mediterranean coast (ca. 500 km), our results show that stable isotope patterns are strongly driven by freshwater discharges [50]. Blue crab signatures were more depleted in δ¹³C and more enriched in δ¹⁵N at low salinities, and contrariwise at high salinities, consistent with findings reported in other ecological studies (e.g., [32–36, 51]) and with observations in dietary resources, particularly for δ¹⁵N. Hence, rather than distance, observed effects of Region and Locality were largely driven by spatial differences in freshwater supply, with Alicante being the region with more oligohaline environments and Murcia the only region with hypersaline sites [40, 52]. For δ¹³C, the effect of salinity is related to a progressive shift in the contributions of organic matter derived from more depleted C3 freshwater macrophytes (e.g., Potamogeton pectinatus in Convenio, Pujol, and Riumar) and phytoplankton (<-25 to -19‰), compared to that derived from more enriched C4 salt-marshes, seagrasses (e.g., C. nodosa in Mar Menor, Alfacs Bay, and the Open Sea), and microalgae in estuarine sites (-18 to -12‰) [31, 32]. In fact, a significant association between δ¹³C and salinity was found for plant resources, whereas for the other dietary groups, the trend was not significant, possibly because they included a pool of different vagile animal species, drifting algae or had low replication (e.g., bivalves). For δ¹⁵N, high signatures have been commonly associated to intensive human uses and eutrophication of waterways [34, 53]. Several of our study localities have been reported to be impacted by agrochemical and/or urban sewage eutrophication leading to serious degradation in the recent literature: the ecological collapse of the Mar Menor lagoon in 2016 [54], long-term alternations of hydrological and trophic conditions in the Ebro Delta and the Albufera lagoon associated to rice agriculture [55, 56], and severe eutrophication and vegetation loss in river basins of the Alicante region [39]. However, based on the capacity of δ¹⁵N to act as an indicator of eutrophication in aquatic ecosystems [57], our results suggest that the degree of the environment degradation is the highest in Alicante (16.4‰) followed by Valencia and Catalunya (13.6 and 12.7‰, respectively), and finally by Murcia (8.0‰) localities. In fact, the association of blue crab δ¹⁵N with salinity levels was even higher than for δ¹³C (by 1.46 times), and significant associations were also found for sediments, plants, bivalves, and even fish, supporting that it is a good approach to the estimation of wastewater inputs. Drifting algae and small crustaceans (amphipods and isopods) also associated with drifting vegetation were the only dietary groups (N≥4) that did not display an association with salinity.

Patterns of isotopic niche were consistent with ANOVA results for both isotopes, and with the effect of the salinity gradient. Spatial overlaps among standard ellipse areas corrected for small sample size (SEAc), indicated Murcia featuring no overlap with other regions except for Catalonia (only 5%), and values from 12% (Catalonia with Alicante) to 26% (Catalonia with Valencia) were observed among the other regions. Yet, in the presence of strong environmental gradients, these differences might be due to feeding at different trophic levels. In contrast, results of relative contributions of food resources to blue crab diet at each Locality from MixSiar did not follow the same patterns of spatial aggregation than those of the δ¹³C and δ¹⁵N, suggesting some possible caveats in the outputs of model runs associated to various factors. First, dietary assessment was conducted with food resources collected only once, but that might feature significant variation in seasonal isotopic signatures as reported for organic matter sources, macrophytes, invertebrates with a short life cycle, and foraging fish [50, 53]. Additionally, although the model points to an overall larger contribution of fish, algae and crustaceans (25.7, 18 and 15.1%, respectively), particularly at certain regions (e.g., 42 to 92% of fish in Alicante), other resources might have been locally available in the shorter term and unaccounted at the moment of sampling. Second, the enrichment factor of blue crab under natural field conditions may only be approximated, since it can change considerably with the type of diet (e.g., [42, 44, 58], and the species features a remarkable dietary plasticity depending on resource availability [16, 17]. Trophic position, however, can provide a robust diagnostic performance of the trophic ecology of the species, but adequate estimation relies on the availability of primary consumers such as bivalves [3, 16, 45, 46, 49], which in our study were limited to 4 of the 11 localities. According to our results, the Open Sea locality adjacent to the Albufera Lagoon (Valencia) is the site featuring the highest TP (3.63), whereas values ≤3, were observed in the other three localities (Alfacs, Encanyissada, and Seco Grande). Despite a limited number of localities with TP, patterns were neither consistent with differences in isotopic niche (the Open Sea in Valencia was more distinctive than Seco Grande in Murcia) or MixSiar results displaying Polychaeta (deposit-feeding species among others, with a TP of ca. 2; [59, 60]) as the main dietary item in the Open Sea (45%). Nevertheless, enhanced TP in the open sea of Valencia, is consistent with a large TP of 4.40 recently reported for the Open Sea of the Ebro Delta (similar also to other local predatory fish; Prado et al., 2024), suggesting that blue crabs in open waters have access to higher-order consumers than in confined waters, estuarine, or riverine environments. Additionally, current values of TP appear significantly lower (ca. 9 to 16%) than those reported in previous estimates in the Alfacs Bay and adjacent coastal lagoons [16], suggesting a progressive decrease in the availability of higher quality preys. Overall, isotopic niche analysis provided a good indication of aquatic ecosystem type and its inherent variability (i.e., trophic niche width), whereas the TP estimated with a baseline standardization that removes the influence of salinity allowed for certain comparison across ecosystems and attained similarly lower values in confined and semi-confined waters than in the open sea [16, 49].

Subadult, immature blue crab sizes were only captured in six out of the eleven study localities. Four of them (those in Catalonia and Pujol in Valencia, in which all sexes and sizes were present), showed significantly more enriched δ¹³C signatures in subadult sizes compared to large individuals, and similar higher values were also observed for δ¹⁵N in Alfacs and Riumar, which could be attributed to ontogenic differences in habitat use and access to food resources. In a previous study by Hoeinghaus and Davis [61], authors showed that the δ¹³C signature of blue crabs ranging from 35 to 165 mm carapace width increased significantly with body size due to greater assimilation of carbon ultimately derived from saltmarsh C4 plants. Similar results were also found by [37, 62] who found that juveniles got enriched in δ¹³C as they age and undergo a gradual shift from a planktonic to a benthic-based diet in marsh areas with Spartina alterniflora. This meshes well with juvenile blue crabs often being abundant in shallow oligohaline and tidal freshwater marshes, which also appear to offer certain osmotic advantage for enhanced molt increments [63] and refuge from predators [64]). Yet, since individuals >70 mm are indicated to have diets similar to adult sizes [15], the absence of significant differences in δ¹³C signatures is also plausible, although this was observed in the two localities with an incomplete replication (Perelló in Valencia and Seco Grande in Murcia). In contrast, higher δ¹³C values (2.6 to 3.5‰ in Catalonia, and 2.4‰ in Pujol, Valencia) in smaller sizes differ from previous literature trends, and suggest that subadults captured in these localities have spent longer time in higher salinity habitats feeding on more enriched food sources. Although early blue crab instars of 2–3 mm have been detected in the Alfacs Bay (unpublished data) and in Valencian sites using larval collectors [25], the proportion of those arriving at larger stages is unknown and could contribute to significantly more enriched signatures in subadults (although more consistently for δ ¹³C than for δ¹⁵N). In the Ebro River, recurrent recent droughts [65] may have increased the extent of the salt wedge thus facilitating the arrival of more enriched juveniles from the open sea. In the Alfacs Bay, summer salinity patterns become more diverse [66] due to the effect of rice cultivation discharges, so juveniles from other areas of the bay could have been attracted to our sampling site, and a similar pattern might have been at play in the Encanyissada lagoon. In contrast, in Pujol, an area of the Albufera lagoon connecting with the open sea through a short drainage channel, oligohaline conditions are driven by flood gates preventing the entrance of seawater, that might also control the entrance of small sized crabs from the open sea. Further SIBER results indicated an overlap of 32% between isotopic niches, that support the undergoing of certain segregation between both investigated size classes. Additionally, 20% higher values of TP were also detected in subadults from the Alfacs Bay, in agreement with much higher values reported for mature females in the Open Sea (TP of 4.4; [49]), and the possible partial retention of the signals.

No significant effects of sex were observed for δ¹³C and δ¹⁵N at any single locality. Yet, for δ¹³C, there were inconsistent differences between large individuals of both sexes; large females showed more enriched signatures in Riumar, similar values to males in Encanyissada and Alfacs (Catalonia), Perelló and Pujol (Valencia), and Cola del Rio and Orones (Alicante), and more depleted signatures in Convenio (Alicante). Additionally, in the open sea (Valencia), only mature females were available, whereas they were absent in Mar Menor (Murcia) at the moment of sampling (but see [67]). Overall, these reduced patterns of differences are consistent with the large overlap in the isotopic niche between sexes (44%), and with the similar TP found for large individuals in the Encanyissada lagoon (M: 2.55 vs. F: 2.31) and in the Alfacs Bay (M: 2.27 vs. F: 2.28). Riumar, in the lower Ebro River (around 3 to 13 km from the mouth), might constitute one of the more similar environments to native areas (relatively large estuary with ample connectivity with the open sea) available in the Spanish Mediterranean. The salt wedge of the Ebro River stretches for ca. 27 km upstream [68], and allows for the distribution of organisms by salinity strata. Also, the salt wedge might favor the return of some females from the open sea with higher isotopic signatures [49], since some individuals can live up to 3 years, depending on environmental conditions and the health of the crab [24]. Additionally, salt wedge increase following recurrent recent droughts [65] (CHEbro, 2023) might have also favored the arrival of females. A somewhat unexpected dominance of large females (N = 17 individuals, whereas only 3 large males featuring similar isotopic patterns could be captured) was observed in Cola del Río located in the mouth of the Segura River (Alicante) at a salinity of around 5, which could be attributed to the proximity to the open sea (about 100 m) and to significant agricultural water discharge at the moment of sampling. Abundance patterns skewed towards the dominance of mature females in areas close to the open sea are commonly reported for native areas (e.g., [23, 69] and have also been observed in the Ebro Delta and in front of the Albufera in Valencia ([49]; this study). Yet, mature females do not seem to be equally attracted by areas of hypersaline waters (>40) areas such as the Mar Menor, as suggested by the absence of megalopae larvae in different years sampled with bongo nets and larval collectors (unpublished data), and largely variable sex ratios observed among quarters [67]. Large males usually dominate in extremely isolated freshwater localities such as the Orones channel (>10 km from the sea) because they do not migrate directionally along the salinity gradient [15]. Only in the Convenio channel connecting the Segura River with the Hondo lagoon, δ¹³C signatures of mature females were lower than those of large males, suggesting that few of them are able to reach the locality from other more oligohaline environments through the irrigation network. However, most of the localities featuring less extreme connectivity in terms of isolation and/ or distance to the sea did not show significant differences in isotopic and abundance patterns by sex, pointing to a more homogeneous environment with regards to salinity [20], particularly in deeper waters (> 0.75 m; [66]).

To conclude, low salinities, often caused by agricultural freshwater discharge and associated eutrophication was a central variable shaping the stable isotope composition (both δ¹³C and δ¹⁵N) of blue crab and dietary resources (mostly δ¹⁵N, except for δ¹³C in plants) along the Spanish Mediterranean coast. Its effect was mostly detected at the spatial scale of region and locality, featuring contrasting salinities (particularly between Murcia and Alicante) whereas individual aspects of sex and size displayed comparably lower effects. Compared to typical distances travelled in and out of estuaries by early stages and gravid females, which can exceed 200 km [23], the most remote locations in Alicante were only 10–15 km from the sea and displayed greater changes and clustering in isotopic values than expected given the short distances. However, entrance to estuarine habitats may be affected by human activities such as the presence of regulated channels, elevated agricultural drainage flow, or reduced river discharge increasing the entrance of the salt wedge, which occurred in most of our study areas and may lead to enhanced isotopic signatures in smaller individuals compared to previous studies ([61, 62] vs. this study). Additionally, confined and semi-confined water masses might display more homogeneous salinity features [20], thus concealing sex patterns between large individuals. Overall, we found a comprehensible agreement between stable isotope patterns across the salinity gradient, and isotopic niche assessment. However, dietary evaluation with stable isotope mixing models often provided wide credible intervals and ambiguous results due to the complex nature of isotopic and fractionation data (see also [70]. For instance, [71] demonstrated the limitations of using stable isotope mixing models to accurately determine the diet of Arctic Peregrine Falcon nestlings, showing that camera monitoring provided more precise diet estimates compared to isotope analysis alone. When possible, we advise the estimation of TP as a reliable alternative for comparatively assessing the local use of food resources [30]. For blue crab, more time-consuming, traditional methods such as stomach contents analyses still provide detailed information about dietary items [72] and could be used to verify results derived from mixing models.

Supporting information

S1 Dataset. Diet species at each locality.

https://doi.org/10.1371/journal.pone.0313429.s001

(XLSX)

S2 Dataset. Ellipses overlap by factor.

https://doi.org/10.1371/journal.pone.0313429.s002

(XLSX)

Acknowledgments

We thank IRTA technicians Lluis Jornet, David Mateu, Lluis Matamoros, and Pau Sa for their help during fieldwork conducted in the Ebro Delta. We would also like to thank the two anonymous reviewers for their comments and suggestions that helped to improve the manuscript.

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Citation: Prado P, Català I, Alcaraz C, Barberà MdC, Guijarro-García E, Falco S (2025) Salinity patterns and local migration determine the isotopic composition of the invasive blue crab, Callinectes sapidus, along the Spanish Mediterranean coast. PLoS ONE 20(2): e0313429. https://doi.org/10.1371/journal.pone.0313429

About the Authors:

Patricia Prado

Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

E-mail: [email protected]

Affiliations: Instituto de Investigación en Medio Ambiente y Ciencia Marina (IMEDMAR-UCV), Universidad Católica de Valencia SVM, Calpe, Alicante, Spain, Institut d’Estudis Professionals Aqüícoles i Ambientals de Catalunya (IEPAAC), La Ràpita, Tarragona, Spain, IRTA La Ràpita, Tarragona, Spain

ORICD: https://orcid.org/0000-0002-4986-2010

Iraida Català

Roles: Data curation, Formal analysis, Methodology, Software, Writing – review & editing

Affiliation: IRTA La Ràpita, Tarragona, Spain

Carles Alcaraz

Roles: Data curation, Formal analysis, Methodology, Software, Validation

Affiliation: IRTA La Ràpita, Tarragona, Spain

ORICD: https://orcid.org/0000-0002-2147-4796

Maria del Carmen Barberà

Roles: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – review & editing

Affiliation: Facultad de Ciencias, Universidad de Alicante, Sant Vicent del Raspeig, Alicante, Spain

Elena Guijarro-García

Roles: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – review & editing

Affiliation: Instituto Español de Oceanografía, Centro Oceanográfico de Murcia, Lo Pagan, Murcia, Spain

Silvia Falco

Roles: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing

Affiliation: Instituto de Investigación para la Gestión Integrada de Zonas Costeras (IGIC), Universitat Politècnica de València, Gandía, Valencia, Spain

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