Crab Carcinus Aestuarii Research Articles

Effects of Bisphenol A analogues and their mixture on the crab Carcinus aestuarii: Cytotoxicity, oxidative stress and damage, neurotoxicity, physiological responses, and bioaccumulation

Bisphenol A (BPA) analogues are emerging contaminants, whose ecotoxicological profile for aquatic species, particularly marine ones, is little known. In this study, the effects of an environmentally realistic concentration (300 ng/L) of three BPA analogues (BPAF, BPF, and BPS) - alone or as a mixture (MIX) – were evaluated for the first time on the crab Carcinus aestuarii. A multibiomarker approach was adopted to assess the effects of 7 and 14 days of exposure on haemolymph parameters, gill and hepatopancreas biochemical parameters, and physiological responses of crabs. Bioaccumulation of the three bisphenols was also investigated in crabs by UHPLC-HRMS. A significant reduction in total haemocyte counts was recorded in crabs exposed for 7 days to BPAF and MIX and for 14 days to the MIX, whereas an increase was found in crabs treated for 14 days with BPAF. Cell proliferation increased significantly in crabs exposed for 14 days to BPS and MIX. An imbalance of the antioxidant system, as well as oxidative damage, was recorded in gills and hepatopancreas. No neurotoxic effects were observed in crabs. At the physiological level, exposure to MIX increased the respiration rate of crabs. As for bioaccumulation, only bisphenol AF was detected in crabs. Overall, the present study demonstrated that BPA analogues can affect some important cellular parameters, induce oxidative stress and alter physiological responses in crabs.

A multibiomarker approach to assess the effects of a BPA analogue-contaminated diet in the crab Carcinus aestuarii

Bisphenol A analogues are largely used plasticisers that are going to replace bisphenol A in many sectors. Due to this replacement, their discharge and presence in the marine coastal areas are increasing, with unknown consequences for organisms and the trophic chain. This study assessed the effects of three different bisphenols (BPAF, BPF and BPS) - alone or as a mixture - provided via food (exposed clams) to the crab Carcinus aestuarii. First, clams were exposed for two weeks to 300 ng/L of each of the three bisphenols and their mixture (100 ng/L of each) to allow the bioaccumulation of the contaminants in bivalves. Then, crabs were fed for two weeks with BPA analogue-exposed clams, while unexposed clams were used to feed control crabs. After 7 and 14 days, haemolymph, gills and hepatopancreas were collected from crabs to measure a battery of biomarkers indicative of cytotoxicity, oxidative stress and damage, neurotoxicity, physiological performance (respiration and excretion rate) and electron transport system activity. Lastly, bioaccumulation of BPA analogues was assessed by UHPLC-HRMS in crabs. Our findings revealed that BPA analogue-exposed clams were able to alter total haemocyte count, haemocyte size and their proliferation. The activity of immune enzymes, such as phosphatases and phenoloxidase was altered. Moreover, we observed an impairment of antioxidant and detoxifying enzymes like SOD, CAT, GST and GPX activities. Alterations of metabolism-involved enzymes and physiological parameters and increased oxidative damage to macromolecules like proteins, lipids, and DNA were also observed in crabs. Among BPA analogues, only bioaccumulation of BPAF, which has the highest Logkow value among the tested bisphenols, was evidenced in crabs. Overall, the obtained results indicated that crabs, under the tested experimental conditions at least, underwent alterations in cellular, biochemical and physiological responses following a diet of bisphenol-exposed clams, suggesting a potential ecotoxicological risk in the marine food chain.

Holistic assessment of dimethoate toxicity in Carcinus aestuarii's muscle tissues.

Dimethoate (DMT) is one of the most harmful and commonly used organophosphate pesticides in agricultural lands to control different groups of parasitic insects. However, this pesticide is considered a dangerous pollutant for aquatic organisms following its infiltration in coastal ecosystems through leaching. Yet, our investigation aimed to gain new insights into the toxicity mechanism of DMT in the muscles of the green crab Carcinus aestuarii, regarding oxidative stress, neurotransmission impairment, histological aspects, and changes in lipid composition, assessed for the first time on the green crab's muscle. Specimens of C. aestuarii were exposed to 50, 100, and 200µg DMTL-1 for 24h. Compared to the negative control group, the higher the DMT concentration, the lower the saturated fatty acids (SFA), and the higher the monounsaturated fatty acids (MUFA). The significant increase in polyunsaturated fatty acid n-6 (PUFA n-6) was related to the high release, mainly, of linoleic acid (LA, C18: 2n6) and arachidonic acid (ARA, C20: 4n6) levels. Biochemical biomarkers showed that DMT exposure promoted oxidative stress, highlighted by increased levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), advanced oxidation protein product levels (AOPP), and protein carbonyl (PCO). Furthermore, the antioxidant defense system was activated, as demonstrated by the significant changes in the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) levels associated with an adaptation process of C. aestuarii to cope with the DMT exposure. This pesticide significantly impairs the neurotransmission process, as evidenced by the inhibition of acetylcholinesterase (AChE) activity. Finally, several histopathological changes were revealed in DMT-treated crabs, including vacuolation, and muscle bundle loss.This research offered new insights into the toxic mechanism of DMT, pointing to the usefulness of fatty acid (FA) composition as a sensitive biomarker in littoral crabs.

Alteration of redox status and fatty acid profile in gills from the green crab (Carcinus aestuarii) following dimethoate exposure

Dimethoate is a broad-spectrum organophosphate insecticide and acaricide. Through various pathways, such as runoff and drift, dimethoate can reach marine environment, and easily impact common organisms in coastal areas, close to agriculture lands, namely crustaceans. The purpose of this study was to investigate the potential effects of dimethoate exposure (50, 100, and 200 μg/l), for 1 day, on a wide range of markers of oxidative stress and neurotransmission impairment, as well as fatty acids composition and histopathological aspect in the gills of the green crab Carcinus aestuarii. A significant increase in n-3 polyunsaturated fatty acids series, namely the eicosapentaenoic acid (C20: 5n3) and its precursor alpha-linolenic acid (C 18: 3n3) in dimethoate-treated crabs was recorded. Concerning n-6 polyunsaturated fatty acids, we noted a high reduction in arachidonic acid (C20:4n-6) levels. Dimethoate exposure increased the levels of hydrogen peroxide, malondialdehyde, lipid hydroperoxides, protein carbonyl, and caused the advanced oxidation of protein products along with enzymatic and non-enzymatic antioxidant-related markers. Acetylcholinesterase activity was highly inhibited following exposure to dimethoate in a concentration-dependent manner. Finally, deleterious histopathological changes with several abnormalities were noted in exposed animals confirming our biochemical findings. The present study offered unique insights to establish a relationship between redox status and alterations in fatty acid composition, allowing a better understanding of dimethoate-triggered toxicity.

Differential feeding rates of native and alien predators on the invasive Asian date mussel Arcuatula senhousia (Benson in cantor, 1842) in a Mediterranean coastal lagoon

AbstractThe alien Asian date mussel Arcuatula senhousia inhabits several coastal environments worldwide. This species can form dense mats where individuals attach through byssus threads, thus altering the structure, functioning and biodiversity of the native communities. We investigated the feeding preference of different predators on this alien species in a Mediterranean coastal lagoon. To do this, a mesocosm experiment was conducted using two crab species (the Mediterranean green crab Carcinus aestuarii) and the Say mud crab Dyspanopeus sayi, and one gastropod, the banded dye‐murex Hexaplex trunculus as predators of A. senhousia with or without byssus mats. Our data suggest that C. aestuarii is a more effective predator against A. senhousia than D. sayi, and that H. trunculus is almost ineffective. A possible implication of this result is the potential use of the native crab C. aestuarii for limit the formation of the byssus mats, thus mitigating their potential negative effects on the native communities.

Identification and characterisation of crustacean hyperglycaemic hormone (CHH) from Mediterranean shore crab Carcinusaestuarii

Identification and characterisation of crustacean hyperglycaemic hormone (CHH) from Mediterranean shore crab Carcinusaestuarii

Color-specific porosity in double pigmented natural 3d-nanoarchitectures of blue crab shell

3D-engineered nano-architectures with various functionalities are still difficult to obtain and translate for real-world applications. However, such nanomaterials are naturally abundant and yet wasted, but could trigger huge interest for blue bioeconomy, provided that our understanding of their ultrastructure-function is achieved. To date, the Bouligand pattern in crustaceans shell structure is believed to be unique. Here we demonstrated that in blue crab Callinectes sapidus, the 3D-nanoarchitecture is color-specific, while the blue and red-orange pigments interplay in different nano-sized channels and pores. Thinnest pores of about 20 nm are found in blue shell. Additionally, the blue pigment co-existence in specific Bouligand structure is proved for the green crab Carcinus aestuarii, although the crab does not appear blue. The pigments interplay, simultaneously detected by Raman spectroscopy in color-specific native cuticles, overturns our understanding in crustaceans coloration and may trigger the selective use of particular colored natural nanoarchitectures for broaden area of applications.

Microplastic in wild populations of the omnivorous crab Carcinus aestuarii: A review and a regional-scale test of extraction methods, including microfibres

Microplastic (MP) has become ubiquitous in the marine environment. Its threat to marine organisms has been demonstrated under laboratory conditions, yet studies on wild populations still face methodological difficulties. We reviewed the methods used to separate MP from soft animal tissues and highlighted a lack of standardised methodologies, particularly critical for synthetic microfibres. We further compared enzymatic and a potassium hydroxide (KOH)-based alkaline digestion protocols on wild crabs (Carcinus aestuarii) collected from three coastal lagoons in the north Adriatic Sea and on laboratory-prepared synthetic polyester (PES) of different colour and polypropylene (PP). We compared the cost-effectiveness of the two methods, together with the potential for adverse quantitative or qualitative effects on MP that could alter the capability of the polymers to be recognised via microscopic or spectroscopic techniques. Only 5.5% of the 180 examined crabs contained MP in their gastrointestinal tracts, with a notably high quantitative variability between individuals (from 1 to 117 particles per individual). All MP found was exclusively microfibres, mainly PES, with a mean length (±SE) of 0.5 ± 0.03 mm. The two digestion methods provided comparable estimates on wild crabs and did not cause any visible physical or chemical alterations on laboratory-prepared microfibres treated for up to 4 days. KOH solution was faster and cheaper compared to the enzymatic extraction, involving fewer procedural steps and therefore reducing the risk of airborne contamination. With digestion times longer than 4 days, KOH caused morphological alterations of some of the PES microfibres, which did not occur with the enzymatic digestion. This suggests that KOH is effective for the digestion of small marine invertebrates or biological samples for which shorter digestion time is required, while enzymatic extraction should be considered as alternative for larger organisms or sample sizes requiring longer digestion times.

Benthic organisms as ecological indicators for the status assessment of coastal ecosystems

The presence of potentially toxic elements (PTE) was determined in different tissues of five selected marine organisms. The As, Cd, Cu, Mn, Mo, Ni, Pb and Zn concentrations were measured in the seagrassCymodocea nodosa, the green algaCladophora echinus, the red algaGelidiella lubrica, the marine topshellPhorcus turbinatusand the littoral crabCarcinus aestuarii, as well as in seawater from Makirina Bay. The levels of As, Cd, Cu, Mn and Zn in the biota were found to exceed those in previously analysed sediments, indicating the bioaccumulation of these PTE. The biota-sediment accumulation factor (BSAF) and concentration factor (CF) varied among different organisms. As regards the five selected species,C. nodosa,C. echinusandG. lubricaproved to be the strongest accumulators of Mn, whileP. turbinatusandC. aestuariishowed a high capacity to accumulate As, Cd, Cu and Zn. These species can be considered as good ecological indicators in the assessment of PTE pollution in marine littoral environments.

Concordant patterns of mtDNA and nuclear phylogeographic structure reveal Pleistocene vicariant event in the green crab Carcinus aestuarii across the Siculo-Tunisian Strait

This study focuses on the population genetic structure of the green crab Carcinus aestuarii along part of the African Mediterranean coast, with the main target to confirm genetic subdivision across the well documented genetic boundary of the Siculo-Tunisian Strait. For this purpose, the mitochondrial COI (cytochrome oxidase I) gene and five polymorphic microsatellite loci were analysed in 144 and 120 specimens, respectively. Our results show the existence of two distinct haplogroups, separated by 16 mutational steps and revealed a non random distribution of the genetic variation along the African Mediterranean coast. Dating analyses, based on the use of different molecular clock models and rates, placed the divergence among both haplogroups at 1.91 Myr (95% HPD: 1.11–2.68 Myr) to 0.69 Myr (95% HPD: 0.44–0.98 Myr). This range of divergence time estimation corresponds to the Early Pleistocene. The particular pattern of genetic divergence among Eastern and Western African Mediterranean populations of C. aestuarii, detected by 2-level AMOVA at the mitochondrial level, was consistent with that inferred from microsatellite analysis and suggests a vicariant event in C. aestuarii. Demographic reconstruction, inferred from mismatch distribution and BSP analyses, yielded different patterns of demographic history between both African Mediterranean groups. The distribution pattern of the two haplogroups across the African Mediterranean coast, along with results of Bayesian analysis of genetic structure revealing an intermediate geographic group between the two divergent groups of the African coast, support the hypothesis of a secondary contact between two historically isolated groups. Although this hypothetical contact zone, thought to be located near the Siculo-Tunisian Strait, still needs to be verified, the asymmetric gene flow from Western to Eastern African Mediterranean, as inferred by the results of a MIGRATE analysis, reinforces the previously mentioned results.

Copper Induced Lysosomal Membrane Destabilisation in Haemolymph Cells of Mediterranean Green Crab (Carcinus aestuarii, Nardo, 1847) from the Narta Lagoon (Albania)

Destabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA). Crabs collected in Narta Lagoon, Vlora (Albania) during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT) and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05), indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05) than in control group, demonstrating the presence of stress on the animals. These results showed thatC. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota.

Osmoregulation and salinity-induced oxidative stress: is oxidative adaptation determined by gill function?

Osmoregulating decapods such as the Mediterranean green crab Carcinus aestuarii possess two groups of spatially segregated gills: anterior gills serve mainly respiratory purposes, while posterior gills contain osmoregulatory structures. The co-existence of similar tissues serving different functions allows the study of differential adaptation, in terms of free radical metabolism, upon salinity change. Crabs were immersed for 2weeks in seawater (SW, 37 ppt), diluted SW (dSW, 10 ppt) and concentrated SW (cSW, 45 ppt). Exposure to dSW was the most challenging condition, elevating respiration rates of whole animals and free radical formation in hemolymph (assessed fluorometrically using C-H2DFFDA). Further analyses considered anterior and posterior gills separately, and the results showed that posterior gills are the main tissues fueling osmoregulatory-related processes because their respiration rates in dSW were 3.2-fold higher than those of anterior gills, and this was accompanied by an increase in mitochondrial density (citrate synthase activity) and increased levels of reactive oxygen species (ROS) formation (1.4-fold greater, measured through electron paramagnetic resonance). Paradoxically, these posterior gills showed undisturbed caspase 3/7 activity, used here as a marker for apoptosis. This may only be due to the high antioxidant protection that posterior gills benefit from [superoxide dismutase (SOD) in posterior gills was over 6 times higher than in anterior gills]. In conclusion, osmoregulating posterior gills are better adapted to dSW exposure than respiratory anterior gills because they are capable of controlling the deleterious effects of the ROS production resulting from this salinity-induced stress.

Morphological differentiation among geographically close populations of the green crab Carcinus aestuarii Nardo, 1847 (Brachyura, Carcinidae) from the Tunisian coast

In the present study, we assessed patterns of morphological variation among populations of the Mediterranean green crabCarcinus aestuariiNardo, 1847 from the Tunisian coast. Eleven morphometric traits were measured on 464 specimens from nine sites, and the pattern of morphological variation among sites was assessed using multivariate approach. Non-parametric MANOVA (NPMANOVA) and non-metric Multidimensional Scaling (MDS) analyses revealed statistically significant differences among the studied locations for both sexes proving that natural variability of the measured traits differs clearly between locations. Canonical discriminant analysis (CDA), on the other hand, showed that over 36% of individuals could be assigned to their location of origin on the basis of their morphology. Correct assignment of individuals to their location was much higher (over 63%) when sites were combined into three regional groups and marked morphological differentiation was found in both sexes. A positive correlation was noted between the squared Mahalanobis distances () and the geographic distances for both male and female green crab populations suggesting isolation by distance. The results obtained indicate that morphological divergence among populations of the green crab can occur over shorter distances than expected based on dispersal potential. Possible drivers of the observed pattern of morphological variation are discussed.

Reproductive biology of the mediterranean green crab Carcinus aestuarii Nardo, 1847 (Crustacea, Brachyura, Portunidae) in Homa Lagoon, Aegean Sea, Turkey

Abstract The Mediterranean green crab Carcinus aestuarii Nardo, 1847 (Crustacea, Brachyura, Portunidae) is a common inhabitant of the shallow waters of Homa Lagoon in Izmir Bay, Turkey, in the eastern Mediterranean. Monthly samples were collected in Homa Lagoon from June 2006 to May 2007 using trammel nets, fyke nets, beach seines and fence traps. In total, 608 males and 559 females of C. aestuarii were collected. The highest sex ratio (81–86% females) was recorded between October and December, and in August (58% females). Ovigerous females were obtained only from November to February with the highest ratio of ovigerous females (55%) observed in January 2007. The maturation curve presenting the carapace width showed that 50% of female crabs with the carapace width of 26.84 ±1.58 mm (r= 0.72) were physiologically mature. Regression analyses indicated that the number of eggs was positively correlated with carapace width and wet weight (P &lt; 0.05, in both cases).

Short-term sublethal hypoxia affects a predator-prey system in northern Adriatic transitional waters

Predation intensity depends on factors that affect both the predator’s ability to locate prey as well as defensive responses by prey to approaching predators. The interactive effects of short-term hypoxia and predation were tested on the survival of two bivalves ( Tapes philippinarum and Musculista senhousia) through laboratory experiments using the crab Carcinus aestuarii as predator. We found M. senhousia to be a focal prey of C. aestuarii but, after non-lethal hypoxia, the crabs’ preference for the focal prey was influenced by the presence of the other prey, T. philippinarum. We observed an environmentally-mediated, non-reciprocal indirect interaction between the two prey species, probably caused by differences in specific traits. Identifying the influence of short-term disturbance on predator-prey relationships is critical for predicting the effects of changes in water quality on trophic interactions and food web dynamics in transitional systems.

Cellular and biochemical parameters in the crab Carcinus aestuarii after experimentally-induced stress: Effects of bacterial injection, leg ablation and bacterial injection/leg ablation combination

The effects of bacterial injection, leg ablation and bacterial injection/leg ablation combination on cellular and biochemical parameters of the crab Carcinus aestuarii were evaluated. Total haemocyte count (THC), haemocyte volume, haemocyte proliferation, phenoloxidase (PO) activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) and CFH glucose levels were used as biomarkers of stress in crabs. After 24h following bacterial injection, no significant variation in THC were observed, whereas haemocyte diameter and volume decreased significantly in injected crabs with respect to controls. Haemocyte proliferation and glucose levels increased significantly in injected crabs, whereas no significant variations in PO activity was recorded between control and injected crabs. THC increased significantly one-day after leg ablation, whereas it decreased significantly after 3 and 7days. Haemocyte diameter and volume did not vary significantly between control and de-clawed crabs at each sampling times. In de-clawed crabs, haemocyte proliferation was significantly higher after 7days, whereas glucose and PO activity increased significantly in CFH after 1 and 3days. The bacterial injection/leg ablation combination caused a statistically significant decrease in both THC and haemocyte diameter and volume, whereas increased significantly glucose levels in CFH. Overall, results obtained demonstrated that stress conditions investigated in the present study affected both cellular and biochemical parameters in C. aestuarii. Results also indicated that crabs can cope with differing stressful conditions modulating their immune parameters depending on the kind of stress undergone. Although preliminary, our results may explain – partially at least – the great adaptability and widespread of C. aestuarii out of its native area.

A simple range expansion model of multiple pathways: the case of nonindigenous green crab Carcinus aestuarii in Japanese waters

Forecasting the range expansion of nonindigenous organisms enables effective quarantine and the development of pre-arrival countermeasures, as well as raises public and scientific concerns among the general public. Here, we present an approach to forecasting the range expansion of the nonindigenous green crab Carcinus in Japanese waters, with consideration of the human-mediated shipping and natural dispersal. Two types of shipping, namely, primary transport to Japan via long-distance ocean-going shipping and secondary transport within Japan via short-distance coastal shipping, were considered. The presence and absence of the crab in 50-km shoreline segments were obtained at 5 year intervals from 1985 to 2005, based on observation records by professional and amateur naturalists. Two types of dispersal kernel, namely, conventional logistic regression and a multiplicative immigration model considering multiple pathways explicitly, were compared. The multiplicative immigration model gave significantly better results. Natural dispersal was the most significant factor, in spite of its low expansion rate: secondary transport by coastal shipping was the second. Transport by international ocean-going shipping was not statistically significant, suggesting quite a small immigration probability of the crab by long voyages. Stochastic simulations forecasted that the crab will invade most of the coasts of western Japan along the Pacific Ocean and Seto Inland Sea by 2055, and will become widespread all over the country by 2205. Quarantine scenarios to eliminate transport by vessels revealed that preventing crab transport by domestic coastal vessels may delay the arrival of crabs in Hokkaido and the northern Japan area by 700 years at maximum.

Solution structures of 2 × 6-meric and 4 × 6-meric hemocyanins of crustaceans Carcinus aestuarii, Squilla mantis and Upogebia pusilla

Arthropod hemocyanins (Hcs) are a family of large, high molecular mass, extracellular oxygen transport proteins. They form oligomeric quaternary structures based on different arrangements of a basic 6×75 kDa hexameric unit. Their complex quaternary structures present binding sites for allosteric effectors and regulate the oxygen binding process in a cooperative manner. In order to describe the functional regulation of arthropod Hcs, a detailed description of their quaternary structure is necessary. We have utilized small angle X-ray scattering to characterize the structure of three arthropod Hcs in unperturbed conditions. Two different levels of complexity are evaluated: for the 2×6-meric case, we analyzed the Hcs of the portunid crab Carcinus aestuarii and stomatopod Squilla mantis, while in the case of 4×6-meric structures, we studied the Hc of the thalassinid shrimp Upogebia pusilla. While C. aestuarii Hc presented a structure comparable to other 2×6-meric crustacean Hcs, S. mantis Hc shows a peculiar and quite unique arrangement of its building blocks, resembling a substructure of giant Hcs found among cheliceratans. For U. pusilla, the arrangement of its subunits is described as tetrahedral, in contrast to the more common square planar 4×6-meric structure found in other arthropod Hcs.

First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea, Decapoda)

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3–5 µm in diameter and 30–50 fL in volume, the second was of about 6–12 µm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20±1.7 µm and 272.30±143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94±1.43 µm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38±1.76 µm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88±1.6 µm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25–30 µm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa’s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim’s panoptical staining and Ehrlich’s triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.

Genetic heterogeneity in populations of the Mediterranean shore crab, Carcinus aestuarii (Decapoda, Portunidae), from the Venice Lagoon

Heterogeneity in genetic composition among recruits, mostly due to a large variance in reproductive success mediated by oceanographic processes, has been reported for marine species but is less understood in coastal lagoons' organisms. Temporal genetic variation in natural populations of the Mediterranean shore crab Carcinus aestuarii was quantified over a multi-year sample. A total of 486 adult crabs were collected at eight different sites of the Venice Lagoon during the period 2005–2007 and screened for genetic variation using 11 microsatellite loci. Two additional samples (N = 115) from neighbouring sites, located approximately 100 km North and South to the Venice Lagoon, were included for the sake of comparison. Our results show significant differences in allelic frequencies at the micro-geographic scale of the Venice Lagoon, observed between sites of collection, typologies of habitat, and areas with different class of ecological risk or pattern of hemocyanin expression. However, this pattern was not constant between years, with significant differences observed mainly in 2005 and 2006, but not in 2007. Our results indicate significant temporal differences suggesting the existence of dynamic processes that act on the genetic pool of this species. Although natural selection and gene flow might play a role, we suggest that genetic drift linked to large variation in the reproductive success of individuals is the most probable scenario to explain the local genetic patterns of differentiation in the Mediterranean shore crab. Our study, by providing the first evidence for the existence of genetic differences in this species at the micro-geographic scale, suggests that a better comprehension of the link between reproduction, recruitment and oceanography is critical to understand how colonization and maintenance of genetic variation is achieved in ephemeral and vulnerable environments such as coastal lagoons.