Grapsidae Family (Decapoda, Brachyura), Semi-Terrestrial Crabs, in Ecotoxicology: State of Knowledge and New Molecular Biomarker Dataset to Develop Investigations

Multiple alignment and phylogenetic analyses

The sequence datasets were aligned using the MAFFT software (http://mafft.cbrc.jp/alignment/server). Evolutionary analyses were conducted in MEGA X software (https://www.megasoftware. net). The best evolutionary model was determined for each dataset, and the maximum likelihood and neighbor-joining methods were applied. Thousand bootstrap replications were done as a test of phylogeny. The predictions of the 3D structure of the proteins of P. marmoratus built using the AlphaFold server (https://alphafoldserver. com/about) were established to illustrate the molecular phylogenies.

State of knowledge on the use of grapsids in ecotoxicology

The grapsids are common in aquatic and semi-terrestrial environments, easily identified, possess a large body, are long-lived, have high trophic status, and are sedentary [17,19]. Therefore, they are a good accumulator of pollutants (accumulators) and express defence responses or behaviour and physiological disturbance when they are contaminated and intoxicated (Table 2) [18]. Ecotoxicological studies were developed for four genera: Goniopsis, Grapsus, Metopograpsus and Pachygrapsus.

Table 2:Listing of bibliographic references for all studies used grapsids in ecotoxicology.

Goniopsis: Davanso, et al. [25] evaluated the Glutathione-S Transferase (GST) and Acetylcholinesterase (AchE) activities in the anterior and posterior gills of Goniopsis cruentata (male and female) in Brazil to estimate the environmental quality of estuarine areas of the Fortaleza Metropolitan region showing a modulation of the enzymatic activities and DNA damage in the distinct stations studied. In West Africa, Numbere [26] analysed the heavy metals (Cd, Pb, Zn) and hydrocarbons in the tissues (carapace, claw, gill, gut, intestine, and pincers) of G. pelii collected in polluted mangrove forests. He showed that males accumulated slightly higher hydrocarbons and metals than females. The order of content metals was Zn >Pb >Cd. Enyi, et al. [27] evaluated the metal content in the tissues of G. pelii in Nigeria, indicating higher chromium and zinc content in meat than in egg and carapace. In parallel, they determined the toxic effects of drilling mud (containing heavy metals) by the crab mortality estimation from 24, 48, 72 and 96 hours. Recently, microplastic pollution was determined in the mangrove species G. pulchra in Columbia, revealing 100% of the MPs occurrence in crabs [28]. The average microplastics per individual was 4.84 particles per gram of soft weight (9 particles/individual).

Grapsus: Miao, et al. [29] investigated metal (As, Cd, Cr, Cu, Hg, Pb, Se, Zn) accumulation in Grapsus tenuicrustatus in the Hawaiian archipelago. The crabs showed high concentrations of essential metals (Cu: 245μg/g dry weight, Zn: 232μg/g dry wt) and non-essential metals (As: 52,232μg/g dry wt; Pb:33μg/g dry wt); compared to coral, and fish (excepted to eels); probably concerning their diet. The red rock crab G. grapsus was also used to estimate the metal pollution in the oceanic island of Trindade (Brazil) [30], with an order of metals in muscle: Zn (110μg/g tissues) >Fe >Cu >Al >As >Hg >Se > Mn > Pb >Ni > Cd (<0.42μg/g tissues). This species collected in Sao Pedro and Sao Paulo Archipelago (Brazil) showed persistent organic pollutants (PCBs) [31] levels in tissues; probably linked to atmospheric transport of pollution from the continent.

Metopograpsus: This genus is lowly integrated in ecotoxicology yet. In 2020; microplastic pollution was estimated in M. frontalis from Hong Kong beaches, showing lower MPs accumulation (0.21±0.06 particles/g wet weight) compared to Macrophthalmus convexus (2.59±0.73 particles/g wt), and Austruca lactea (2.84±0.44 particles/g wt) [32].

Pachygrapsus: This genus was recognised as a sentinel to study the bioaccumulation and impacts of inorganic and organic pollutants [33-36]. The relationship between the metal (As, Cd, Pb, and Cu) accumulation and genetic diversity decrease (microsatellite loci) was studied on P. marmoratus originated from the Tuscan coast (Italy). Thus, loss of genetic variability (genetic erosion) was shown in populations inhabiting polluted sites compared to reference sites [33]. The metals, respecting the order Cu >As >Pb >Cd, revealed a differential accumulation in tissues, with higher content in the hepatopancreas (detoxification organ). This species has been used to evaluate the metal pollution on the Italian coast (Rosignano site) to distinct metals (Cr, Cu, Ni, Pb, Zn) [37]. In the context of oil spill pollution, P. transversus, the most representative crab species in Brazilian reefs, was examined to detect the eventual disorder in its abundance, relative growth, and sex ratio. In polluted sites, the size of crabs and the abundance of females decreased drastically [38]. Thus, the analysis of the dynamic population of P. transversus appeared also as a good indicator of environmental disturbance. Therefore, the embryos and larvae may be used in toxicological investigations. For example, it was shown that if the larval spawning of P. marmoratus were exposed to high copper concentrations, high antioxidative (Catalase, SOD) and esterase (BChE, PChE) activities are induced to compensate for the eventual cellular damage [16]. Recently, Caliani, et al. [35] developed an integrated multidisciplinary approach in Pachygrapsus marmoratus including chemical analyses, enzymatic biomarker activities (AchE, Catalase, GST, …), larval development study and genotoxic tests (nuclear abnormality) to detect the impacts of port activities in Italy. They concluded that P. marmoratus is an excellent bioindicator, in the Mediterranean Sea, for monitoring environmental quality because it showed a high accumulation of contaminants (like metals) in tissues, and has a modulation of responses at different levels (respecting the Adverse Outcome Pathways) according to the pollution level. In parallel, the embryos of Pachygrapsus crassipes, abundant in estuaries and beaches in the USA, were used to develop biotests to determine the bioconcentration of the pesticides [20].

Characterization of molecular biomarkers extracted from -omics data for Pachygrapsus marmoratus

Forty-five biomarkers were characterised in the Pachygrapsus marmoratus transcriptome (Table 3, Figure 2). All the predicted sequences included the complete encoding zone (ATG-stop codon), except for Acetylcholinesterase and Hsp90. We identified biomarkers implicated in apoptosis (AIF, APIP, BAG, Bcl2, CAMK, caspase 1-like, caspase 7, caspase 8, FADD, IAP, P53, TNF), the antioxidative system (catalase, Cu/Zn SOD, Mn SOD, GPx, Peroxiredoxin, Thioredoxin), detoxification system (ATP-dependent translocase ABCB1- like, GST, P450), Energy reserves (G6PDH, Glycogen synthase kinase 3, glycogen synthase, LDH; pyruvate dehydrogenase phosphatase- 1), general stress responses (hsp60, hsp70, hsp90, metallothionein), immune system (ATG7, Dorsal, galectin, Inhibitor of nuclear factor kappa-B kinase, JAK, Pelle, Relish, STAT, TAK1, Tube protein), neurotoxicity (Acetylcholinesterase, Carboxylesterase), and reprotoxicity (methyl famesoate epoxidase, MOIH, vitellogenin). We searched the homologous sequences of P. marmoratus biomarkers in other crab species using Blastn and requested a parallel in the nr/nt nucleotide database. The phylogenetic analyses were carried out only when we found many complete sequences in several crab families. Thus, we establish the phylogenetic relationships for caspases, Hsp70, metallothioneins, Peroxiredoxin, Superoxide dismutase (Cu/Zn SOD, Mn-SOD, and vitellogenin (Figures 3-9). The evolutionary analyses based on the metallothionein (mts) sequences showed that P. marmoratus mts (180bp) were logically close to Eriocheir sinensis (Varunidae) included in Grapsoidea (Figure 3). The same topologies were noted in the phylogenetic analyses based on Hsp70 (P. marmoratus: 1953bp) or Vitellogen (P. marmoratu s: 7782bp) with the validation of the sections Thoracotremata and Heterotremata (Figures 4 & 5).

Table 3:Forty-five molecular biomarkers from Pachygrapsus marmoratus were identified in the Transcriptome Shotgun Assembly (TSA) database using blastn (https://blast.ncbi.nlm.nih.gov) and distinct reference sequences. All coding predicted sequence includes ATG-stop codon, except Acetylcholinesterase and Hsp90, which are partial.

Figure 2:Illustration indicating the forty-five biomarkers characterised in the Pachygrapsus marmoratus transcriptome.

Figure 3:Evolutionary analyses of complete cDNA metallothioneins in crabs (21 sequences) using the Maximum Likelihood method and Kimura 2-parameter+G model (MEGA X software). The tree with the highest log likelihood (-1652.36) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter=0.4762)). The bootstrap values (1000 replications), superior to 50%, are indicated (ML/ NJ).

Figure 4:Phylogenetic analyses based on complete nucleotide Hsp70 sequences of crabs (18 sequences), using MEGA X (Maximum Likelihood method, Tamura-Nei model). The gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.3031)). The tree with the highest log likelihood (-9088.00) is shown. The bootstrap values (1000 replications), superior to 50%, are indicated (ML/NJ).

Figure 5:Phylogenetic tree based on Vitellogenin cDNA for crabs (15 coding complete sequences), using MEGA X (Maximum Likelihood method and Hasegawa-Kishino-Yano model) [1]. The tree with the highest log likelihood (-49889.11) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter=2.1642)). The rate variation model allowed some sites to be evolutionarily invariable ([+I], 11.86% sites).

Figure 6:Phylogenetic tree based on Vitellogenin cDNA for crabs (15 coding complete sequences), using MEGA X (Maximum Likelihood method and Hasegawa-Kishino-Yano model) [1]. The tree with the highest log likelihood (-49889.11) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter=2.1642)). The rate variation model allowed some sites to be evolutionarily invariable ([+I], 11.86% sites).

Figure 7:Phylogenetic analyses of the Cu/Zn SOD in crabs (11 complete sequences) using the Maximum likelihood method (Kimura-2 parameter model +G, parameter=3.4322; +I: 12,94% sites). The tree with the highest log likelihood (-4946.62) is shown. The bootstrap values (1000 replications), superior to 50%, are indicated (ML/NJ).

Figure 8:Phylogenetic analyses of the Mn-SOD in crabs (33 complete sequences) using the Maximum likelihood method (Kimura-2 parameter model +G, parameter = 0.5603). The tree with the highest log likelihood (-9454.32) is shown. The topology revealed the cytosolic and mitochondrial Mn-SOD groups. The bootstrap values (1000 replications), superior to 50%, are indicated (ML/NJ)..

Figure 9:The evolutionary history of caspases was inferred by using the Maximum Likelihood method and the Hasegawa-Kishino-Yano model. The tree with the highest log likelihood (-13368.41) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter =2.4550)). This analysis involved 15 nucleotide sequences.

The Peroxiredoxin (Prx) relationship revealed that the sequence isolated to Pachygrapsus marmoratus (597 bp) is probably a Prx1 isoform (Figure 6). The Cu/Zn SOD phylogeny established the existence of two groups and the P. marmoratus sequence (627bp) is a Cu/Zn SOD1 (Figure 7). Two groups were also noted for Mn- SOD (cytosolic and mitochondrial), the P. marmoratus cDNA (861 bp) was identified as a cytosolic isoform (Figure 8). Three types of caspase were characterised in P. marmoratus (caspase 1: 972bp, caspases 7: 1050bp, and caspase 8: 1488bp) (Figure 9). The datasets of the complete coding sequences for the other biomarkers were insufficient to build a molecular phylogeny. Effectively, many biomarkers were available for a few species (Table 4). The most complete sequences of biomarkers in the TSA and nr/nt NCBI databases were those found of Eriocheir sinensis (Varunidae, Grapsoidea), Portunus trituberculatus and Scylla paramamosain (Portunidae, Portunoidea). This research has revealed that -the omic data of crabs is very poorly studied, and that it will be primordial to establish transcriptomes and genomes of distinct species to isolate biomarker sequences and develop molecular ecotoxicological research on crabs.

Table 4:List the biomarkers found in distinct transcriptomes of brachyurans, using Blastn program (https://blast. ncbi.nlm.nih.gov) in the TSA database and requests in the nr/nt NCBI database. Many homologous complete sequences have been found in Eriocheir sinensis (Varunidae, Grapsoidea), Portunus trituberculatus and Scylla paramamosain (Portunidae, Portunoidea).

The crabs are predator/scavenger species, playing a primordial function in aquatic ecosystems. Their abundance, species diversity, large size and high accumulation of pollutants (allowing studies on differential bioconcentration of pollution in tissues), led to consider brachyuran crabs as a biomonitoring tool [39]. For example, distinct species are considered sentinel species: Carcinus maenas (Portunidae) [40,41], Macrophthalmus spp. (Macrophthalmidae) [42-44], Portunus spp. (Portunidae) [45,46], Scylla serrata (Portunidae) [47-49], and Ucides cordatus (Ucididae) [50]. Other species are also used in ecotoxicological investigations, such as Callinectes amnicola (Portunidae) [51], Callinectes sapidus [52], Carcinus estuarii (Portunidae) [53,54], Eriocheir sinensis (Varunidae) [55,56], Neohelice granulata (Varunidae) [57], and Ocypode quadrata (Ocypodidae) [58]. We have admitted that many species used in ecotoxicology are included in the Portunidae family (Heterotremata section). Thus, surprisingly, the grapsids (Table 1 & Figure 1), semi-terrestrial crabs, abundant along the coats and mangroves, recognised as good accumulators of pollutants, have been underestimated in ecotoxicology as model species. A putative explanation of this fact is a low number of sequences of biomarkers characterised and submitted in international databases. The ecotoxicological investigations have been focused on pollution accumulation and sometimes antioxidative responses (Table 2). Inside Grapsidae, Pachygrapsus genus has a larger geographical distribution (Atlantic and Indo-Pacific Oceans, the Mediterranean, and the Black Seas) and it includes more species (12 sp.) compared to other genera (Geograpsus Stimpson, 1858: 4 species; Goniopsis De Haan, 1833: 3 sp.; Grapsus Lamarck, 1801: 8 sp.; Leptograpsus H. Milne Edwards, 1853: 1 sp.; Metopograpsus H. Milne Edwards, 1853: 1 sp.; and Planes Bowdich, 1825: 2 species) [3]. Among the four genera used in ecotoxicology (Goniopsis, Grapsus, Metopograpsus, Pachygrapsus), Pachygrapsus appeared to be the most studied (Table 2). Inside this genus, P. marmoratus was more analysed compared to P. crassipes and P. tranversus (Table 2). Therefore, the unique transcriptome obtained from grapsids was for Pachygrapsus marmoratus. In this case, to fill the gap of molecular information about biomarkers in grapsids, we have characterised 45 biomarkers in the -omics data of Pachygrapsus marmoratus (Grapidae) (Table 3). Our investigation allowed identify the complete coding sequences of biomarkers involved in (antioxidative response, detoxification system, energy reserves, general stress, immunity, neurotoxicity, and reprotoxicity) (Figure 2). The datasets of crabs, which have been sources of complete coding biomarker sequences matching with the P. marmoratus data were Eriocheir sinensis (Varunidae) Portunus trituberculatus (Portunidae), Scylla paramamosain (Portunidae) (Table 4). Several transcriptomes and isolated biomarker sequences were effectively available for these species. Unfortunately, few datasets of biomarker sequences were sufficiently completed to establish molecular phylogenies. Thus, we built evolutionary analyses to caspases, Hsp70, metallothioneins, Peroxiredoxin, Superoxide dismutase (Cu/Zn SOD, Mn-SOD), and vitellogenin (Figures 3-9). The molecular phylogenies confirmed the identity of the complete sequences characterised in P. marmoratus and the relationship between the Grapsoid sequences. We suggested that the P. marmoratus dataset can allow gene expression or molecular evolution investigations in future. It may be used to develop ecotoxicological investigations in Pachygrapsus marmoratus. For example, we will carry out a gene expression study on P. marmoratus collected in France (North-East Atlantic Ocean) and in Tunisia (The Mediterranean Sea) to compare with the same experimental conditions (exposure to the same pollutants: metals, pesticides) if two distinct geographical isolates will express the same levels of stress responses (antioxidative responses, detoxification system, energy reserves, general stress, neurotoxicity, and reprotoxicity).

Our dataset of P. marmoratus sequences could promote the use of the grapsids in ecotoxicology because it could be used to characterise similar biomarkers in other Pachygrapsus spp. such as P. transversus along the coasts of Africa and America; or P. minutus living in the Indo-Pacific and Pacific oceans, or to identify by the design of degenerated primers, defined after a multiple alignment of grapsoids (E. sinensis, P. marmoratus) sequences in the grapsids.

1. Macleay WS (1938) Illustrations of the Annulosa of South Africa: Being a portion of the objects of natural history chiefly collected during an expedition into the interior of South Africa under the direction of Dr. Andrew Smith in the years 1834, 1835 and 1836;. Smith, Elder and Company, UK.
2. Cannicci S, Paula J, Vannini M (1999) Activity pattern and spatial strategy in Pachygrapsus marmoratus (Decapoda: Grapsidae) from Mediterranean and Atlantic shores. Mar Biol 133: 429-435.
3. Ng PK, Guinot D, Davie PJ (2008) Systema Brachyurorum: Part I. An annotated checklist of extant Brachyuran Crabs of the world. Raffles Bull Zool 17: 1-286.
4. Ip BHY, Schubart CD, Tsang LM, Chu KH (2015) Phylogeny of the shore crab family Grapsidae (Decapoda: Brachyura: Thoracotremata) based on a multilocus approach: Molecular phylogeny of Grapsid Zool J Linn Soc 174(2): 217-227.
5. Wetzer R, Martin JW, Boyce SL (2009) Evolutionary origin of the gall crabs (family Cryptochiridae) based on 16S rDNA sequence data. 1^st (edn.), Decapod Crustacean CRC Press, USA, pp. 1-16.
6. Schubart CD (2011) Reconstruction of phylogenetic relationships within Grapsidae (Crustacea: Brachyura) and comparison of trans-isthmian versus amphi-atlantic gene flow based on mtDNA. Zool Anz 250(4): 472-478.
7. Tsang CTT, Schubart CD, Chu KH, Ng PKL, Tsang LM (2022) Molecular phylogeny of Thoracotremata Crabs (Decapoda, Brachyura): Toward adopting monophyletic superfamilies, invasion history into terrestrial habitats and multiple origins of symbiosis. Mol Phylogenet Evol 177: 107596.
8. Poupin J, Davie PJF, Cexus JC (2005) A revision of the genus Pachygrapsus Randall, 1840 (Crustacea: Decapoda: Brachyura, Grapsidae), with special reference to the Southwest Pacific Species. Zootaxa 1015(1): 1-66.
9. Barros MSFD, Calado TCDS, Araújo MDSLC (2020) Plastic ingestion lead to reduced body condition and modified diet patterns in the rocky shore crab Pachygrapsus transversus (Gibbes, 1850) (Brachyura: Grapsidae). Mar Pollut Bull 156: 111249.
10. Frusher SD, Giddins RL, Smith TJ (1994) Distribution and abundance of Grapsid Crabs (Grapsidae) in a mangrove estuary: Effects of sediment characteristics, salinity tolerances, and osmoregulatory ability. Estuaries 17: 647-654.
11. Lee SY (1998) Ecological role of Grapsid crabs in mangrove ecosystems: A review. Mar Freshwater Res 49(4): 335-343.
12. Robertson AI, Dixon P, Daniel PA (1988) Zooplankton dynamics in mangrove and other nearshore habitats in tropical Australia. Mar Ecol Prog Ser 43(1/2): 139-150.
13. Madeira D, Narciso L, Cabral HN, Diniz MS, Vinagre C (2012) Thermal tolerance of the crab Pachygrapsus Marmoratus: Intraspecific differences at a physiological (CTMax) and molecular level (Hsp70). Cell Stress and Chaperones 17(6): 707-716.
14. Madeira D, Narciso L, Diniz MS, Vinagre C (2014) Synergy of environmental variables alters the thermal window and heat shock response: An experimental test with the crab Pachygrapsus Marmoratus. Mar Environ Res 98: 21-28.
15. Karadal O (2018) Survival rate and restricted growth of marbled crab (Pachygrapsus Marmoratus) in different salinity levels. Ege J Fish Aquat Sci 35(4): 407-416.
16. Oliva M, Marchi LD, Cuccaro A, Casu V, Tardelli F, et al. (2019) Effects of copper on larvae of the marbled crab Pachygrapsus Marmoratus (Decapoda, Grapsidae): Toxicity test and biochemical marker responses. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 223: 71-77.
17. Phillips DJH, Segar DA (1986) Use of bio-indicators in monitoring conservative contaminants: Programme design imperatives. Mar Pollut Bull 17(1): 10-17.
18. Beeby A (2001) What do sentinels stand for? Environ Pollut 112(2): 285-298.
19. Basu N, Scheuhammer AM, Bursian SJ, Elliott J, Rouvinen-Watt K, et al. (2007) Mink as a sentinel species in environmental health. Environ Res 103(1): 130-144.
20. Smalling KL, Morgan S, Kuivila KK (2010) Accumulation of current‐use and organochlorine pesticides in crab embryos from Northern California, USA. Enviro Toxic and Chemistry 29(11): 2593-
21. Xuereb B (2009) Development of markers of neurotoxicity and endocrine disruption in the freshwater Amphipod Gammarus Fossarum. Ecology, Environment, University of Metz, France.
22. Menze MA, Fortner G, Nag S, Hand SC (2010) Mechanisms of apoptosis in Crustacea: What conditions induce versus suppress cell death? Apoptosis 15(3): 293-312.
23. Chen F, Wang K (2019) Characterization of the innate immunity in the mud crab Scylla paramamosain. Fish Shellfish Immunol 93: 436-448.
24. Pinosio S, Fratini S, Cannicci S, Oliva M, Pretti C, et al. (2021) De novo transcriptome assembly for Pachygrapsus Marmoratus, an intertidal brachyuran crab. Mar Genomics 55: 100792.
25. Davanso MB, Moreira LB, Pimentel MF, Costa-Lotufo LV, Abessa DMDS (2013) Biomarkers in mangrove root crab Goniopsis cruentata for evaluating quality of tropical estuaries. Mar Environ Res 91: 80-88.
26. Numbere AO (2019) Bioaccumulation of total hydrocarbon and heavy metals in body parts of the West African Red Mangrove Crab (Goniopsis Pelii) in the Niger Delta, Nigeria. Int Lett Nat Sci 75: 1-12.
27. Enyi IO, Babatunde BB, Hart AI (2020) Heavy metal uptake and bioaccumulation by mangrove grab (Goniopsis Pelli) from used drilling mud, Niger Delta, Nigeria. J Appl Sci Environ Manag 24(3): 477-482.
28. Gallego MAA, Salamanca EJP, Peñaranda MLP, Mora CAQ, Kintz JC (2023) Presence of microplastics in macroinvertebrates of mangroves in the Colombian Pacific: Are feeding behaviors determinant for its abundance? Available at Social Science Research network 4384915.
29. Miao X, Woodward LA, Swenson C, Li QX (2001) Comparative concentrations of metals in marine species from French frigate shoals, North Pacific Ocean. Mar Pollut Bull 42(11): 1049-1054.
30. Theophilo CYS, Ribeiro AP, Trevizani TH, Majer AP, Montone RC, et al. (2022) Assessment of crabs from Trindade, a Brazilian remote Island: Support to marine studies. Mar Pollut Bull 182: 113922.
31. Dias PS, Cipro CVZ, Taniguchi S, Montone RC (2013) Persistent organic pollutants in marine biota of São Pedro and São Paulo Archipelago, Brazil. Mar Pollut Bull 74(1): 435-440.
32. Xu X, Wong CY, Tam NFY, Lo HS, Cheung SG (2020) Microplastics in invertebrates on soft shores in Hong Kong: Influence of habitat, taxa and feeding mode. Sci Total Environ 715: 136999.
33. Fratini S, Zane L, Ragionieri L, Vannini M, Cannicci S (2008) Relationship between heavy metal accumulation and genetic variability decrease in the intertidal Crab Pachygrapsus Marmoratus (Decapoda; Grapsidae). Estuar Coast Shelf 79(4): 679-686.
34. Baratti M, Pinosio S, Gori M, Biricolti S, Chini G, et al. (2022) Differential gene expression and chemical patterns of an intertidal crab inhabiting a polluted port and an adjacent marine protected area. Sci Total Environ 822: 153463.
35. Caliani I, Cannicci S, Pretti C, Baratti M, Contini G, et al. (2022) A multidisciplinary integrated approach using Pachygrapsus Marmoratus to assess the impact of port activities on Mediterranean marine protected areas. Chemosphere 312: 137129.
36. Shaiek M, Zaaboub N, Ayas D, Martins MVA, Romdhane MS (2018) Crabs as bioindicators of trace element accumulation in Mediterranean Lagoon (Bizerte Lagoon, Tunisia). J Sediment Environ 3(1): 1-11.
37. Bonsignore M, Manta DS, Mirto S, Quinci EM, Ape F, et al. (2018) Bioaccumulation of heavy metals in fish, crustaceans, molluscs and echinoderms from the Tuscany Coast. Ecotoxicol Environ Saf 162: 554-562.
38. Santana JL, Rios AS, Calado TCS, Zanardi-Lamardo E, Souza-Filho JF (2022) Reef crab population changes after oil spill disaster reach Brazilian tropical environments. Mar Pollut Bull 183: 114047.
39. Arya S, Trivedi JN, Vachhrajani KD (2014) Brachyuran crabs as a biomonitoring tool: A conceptual framework for chemical pollution assessment. Int Res J Environ Sci 3(1): 49-57.
40. Leignel V, Stillman JH, Baringou S, Thabet R, Metais I (2014) Overview on the European green crab Carcinus Spp. (Portunidae, Decapoda), one of the most famous marine invaders and ecotoxicological models. Environ Sci Pollut Res 21(15): 9129-9144.
41. Rodrigues ET, Pardal MÂ (2014) The Crab Carcinus Maenas as a suitable experimental model in ecotoxicology. Environ Int 70: 158-182.
42. Saher NU, Siddiqui AS (2019) Occurrence of heavy metals in sediment and their bioaccumulation in sentinel crab (Macrophthalmus Depressus) from highly impacted coastal zone. Chemosphere 221: 89-98.
43. Saadati M, Soleimani M, Sadeghsaba M, Hemami MR (2020) Bioaccumulation of heavy metals (Hg, Cd and Ni) by sentinel crab (Macrophthalmus Depressus) from sediments of Mousa Bay, Persian Gulf. Ecotoxicol Environ Saf 191: 109986.
44. Koyama A, Inui R (2024) Sediment temperature characteristics and their relation to distribution patterns of two sentinel crab species in intertidal flats in Western Japan. Sci Rep 14: 861.
45. Pan L, Xu R, Wen J, Guo R (2017) Assessing PAHs pollution in Shandong coastal area (China) by combination of chemical analysis and responses of reproductive toxicity in crab Portunus Trituberculatus. Environ Sci Pollut Res 24(16): 14291-14303.
46. Dghim A, Ameur WB, Annabi A (2023) Assessment of the accumulation of trace metals and oxidative stress response biomarkers in the Portunid Portunus Segnis. Appl Sci 13(12): 7197.
47. Oosterom JV, King SC, Negri A, Humphrey C, Mondon J (2010) Investigation of the mud crab (Scylla Serrata) as a potential bio-monitoring species for tropical coastal marine environments of Australia. Mar Pollut Bull 60(2): 283-290.
48. Baag S, Mandal S (2023) The influence of ocean acidification and warming on responses of Scylla Serrata to oil pollution: An integrated biomarker approach. Comp Biochem Physiol B, Biochem Mol Biol 266: 110847.
49. Valsan G, Tamrakar A, Warrier AK (2024) Microplastics in Scylla Serrata: A baseline study from Southwest India. Mar Pollut Bull 200: 116109.
50. Pinheiro MAA, Souza FVBD, Boos H, Duarte LFDA (2022) Cytotoxicity, genotoxicity, and impact on populations of the mangrove sentinel species, Ucides Cordatus (Linnaeus, 1763) (Brachyura, Ocypodidae) after an environmental disaster at Cubatão, São Paulo, Brazil. Nauplius 30: e2022025.
51. Anyanwu BO, Chris DI (2023) Human health hazard implications of heavy metals concentration in swimming crab (Callinectes Amnicola) from polluted creeks in Rivers State, Nigeria. Case Stud Chem Environ Eng 7: 100325.
52. Çoğun HY, Firat Ö, Aytekin T, Firidin G, Firat Ö, et al. (2017) Heavy metals in the blue crab (Callinectes Sapidus) in Mersin Bay, Turkey. Bull Environ Contam Toxicol 98(6): 824-829.
53. Ricciardi F, Matozzo V, Binelli A, Marin MG (2010) Biomarker responses and contamination levels in crabs (Carcinus Aestuarii) from the Lagoon of Venice: An integrated approach in biomonitoring estuarine environments. Water Res 44(6) 1725-1736.
54. Hayder-Ben YN, Selmi S (2019) Biomarker responses to pollution in the Mediterranean green crab Carcinus Eastuarii living in the Gulf of Gabès (Tunisia). Journal of Materials and Environmental Science 10(1): 45-53.
55. Ni X, Zhou H, Liu Y, Zhan J, Meng Q, et al. (2023) Toxic Effects of tire wear particles and the leachate on the Chinese Mitten Crab (Eriocheir Sinensis). Environmental Pollution 335: 122354.
56. Fang L, Chen X, Fan L, Hu G, Qiu L, et al. (2024) Environment consistently impact on aquaculture: The predominant source of residual pollutants in cultured Chinese Mitten Crab (Eriocheir Sinensis) across China. Heliyon 10(11): e32418.
57. Beltrame MO, Marco SGD, Marcovecchio JE (2011) The burrowing crab Neohelice Granulata as potential bioindicator of heavy metals in estuarine systems of the Atlantic Coast of Argentina. Environ Monit Assess 172(1-4): 379-389.
58. Costa LL, Arueira VF, Costa MF, Beneditto APM, Zalmon IR (2019) Can the Atlantic Ghost crab be a potential biomonitor of microplastic pollution of sandy beaches sediment? Marine Pollution Bulletin 145: 5-13.