Integrating Molecular and Chemical Analyses for Assessing Blue Swimmer Crab Portunus Health and Genetic Diversity in Pakistani Coastal Waters
Saima Majeed1, Salma Javed2, Nazakat Hussain Memon3, Talat Sharafat Rehmani4, Faraz Ahmed Abro5, Muhammad Idrees1 and Asif Inam1
1Department of Maritime Sciences, Bahria University, Karachi Campus, 75260, Pakistan
2National Nematological Research Centre, University of Karachi, Karachi, Pakistan
3Department of Biochemistry, Ghulam Muhammad Mahar Medical College Sukkur, Shaheed Mohtarma Benazir Bhutto Medical University, Larkana, Sindh
4Department of Humanities and Social Sciences, Bahria University, Karachi Campus, 75260, Pakistan
5Department of Zoology, University of Sindh, Jamshoro, Sindh
ABSTRACT
The blue swimming crab species Portunus pelagicus and Portunus segnis represent crucial components of commercial fisheries in the Northern Arabian Sea (NAS). This study employs partial coding regions of the cytochrome oxidase subunit 1 (COI) gene for DNA barcoding, with accession numbers OL840323 and OL840324 assigned to Portunus pelagicus and Portunus segnis, respectively, and deposited in the GenBank database. Analysis revealed high haplotype diversity and low nucleotide diversity within the populations of Portunus spp. Neutrality tests, specifically Tajima’s D and Fu’s F, yielded non-significant results. However, mismatch analysis indicated a potential population expansion event in the Arabian Sea. Evolutionary analyses were conducted comparing 21 sequences of Portunus spp. from GenBank, including 2 from Pakistan and 19 from various other regions, based on COI variation. Results from the analysis of molecular variance (AMOVA) suggested significant phylogeographic structuring (P < 0.05). The study highlights the efficiency of DNA barcoding in species identification, particularly in delineating cryptic varieties. Additionally, seasonal variations in the concentrations of ten trace elements, in carapace meat samples from 210 blue swimming crabs (Portunus pelagicus and Portunus segnis) collected from two locations in Karachi, Pakistan: West Wharf Fish Harbor (n = 100) and Korangi Creek (n = 110). Data collected during monsoon including zinc (Zn), iron (Fe), copper (Cu), cobalt (Co), chromium (Cr) as essential trace elements revealed the following order of essential trace elements: Fe > Zn > Cu > Co > Cr. In contrast, toxic trace elements such as aluminum (Al), lead (Pb), mercury (Hg), arsenic (As), and cadmium (Cd) were found in the order: Pb > Cd > Al > As > Hg and in non-monsoon period essential and toxic elements were found in the order: Fe>Zn>Cu>Cr>Co and As>Pb>Cd>Al>Hg. Metal concentrations were assessed using Atomic Absorption Spectroscopy (AAS), which was chosen for its sensitivity and accuracy in quantifying trace elements. The presence of elevated levels of essential trace elements (Fe, Zn, and Cu) in the aquatic environment is attributed to industrial and maritime activities in the Arabian Sea. Understanding the dynamics of these populations and their trace element uptake is significant for conservation, fishery management, and public health.
Article Information
Received 12 July 2024
Revised 25 December 2024
Accepted 02 January 2025
Available online 18 March 2025
(early access)
Published 26 January 2026
Authors’ Contribution
SM designed and conducted experiments, and collected data. SJ conceived and designed the study. NHM analysed and interpreted the data. TSR contributed to the methodology and validation. FAA performed data analysis and interpretation. MI and AI contributed to the methodology and validation.
Key words
DNA barcoding, Haplotype, Nucleotide, Evolutionary, Cryptic varieties, Maritime
DOI: https://dx.doi.org/10.17582/journal.pjz/20240712071015
* Corresponding author: [email protected], [email protected]
0030-9923/2026/0002-0633 $ 9.00/0
Copyright 2026 by the authors. Licensee Zoological Society of Pakistan.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
INTRODUCTION
The blue swimmer crab, Portunus pelagicus (Linnaeus, 1758), also known as the flower crab, is a significant species in the commercial fisheries sector, valued for its economic and ecological contributions. Found predominantly in the coastal regions of the Indo-Pacific, P. pelagicus, and P. segnis both as a predator and as a prey, are vital to the benthic environment (Maryani et al., 2023). This species is widely distributed along the coastal waters of Pakistan, particularly in the Karachi region, where it supports local fisheries and contributes to the livelihoods of coastal communities (Siddiqui et al., 2008).
Global production exceeded 200,000 tons in 2013 (Noori et al., 2015). These crabs hold both commercial and ecological importance across various regions, including the Northwestern Indian Ocean (NWIO) (FAO, 2016). The blue swimming crab, comprising species such as Portunus pelagicus (Linnaeus, 1758) and Portunus sanguinolentus (Herbst, 1783), among others, is particularly noteworthy. More than 200 crab species, including commercially significant ones like Portunus pelagicus, Charybdis feriata, Portunus sanguinolentus, and Scylla serrata, have been reported in Pakistani waters (Kazmi, 2003), making them suitable for both international trade and local consumption (FAO, 2016).
Traditional taxonomic identification of the blue swimmer crab relies on morphological characteristics such as carapace shape, size, and coloration patterns, which can be variable and subject to misinterpretation, especially among juvenile or geographically distinct populations. This variability underscores the need for robust molecular tools to complement traditional taxonomy and accurately delineate species boundaries. The advent of DNA barcoding, a molecular technique based on sequencing short, standardized gene regions, has revolutionized species identification by providing a reliable method for discriminating between closely related taxa (Saher et al., 2019). By targeting highly conserved regions of the genome, such as mitochondrial DNA, DNA barcoding offers a rapid and cost-effective means of species identification that transcends morphological variation (Joesidawati et al., 2023).
In the case of the blue swimmer crab, DNA barcoding presents an opportunity to overcome taxonomic challenges associated with morphological variability and cryptic speciation. By targeting specific regions of the genome unique to P. pelagicus, such as the cytochrome c oxidase subunit I (COI) gene, researchers can accurately distinguish it from closely related congeners and facilitate the identification of mislabeled or adulterated seafood products in the market. In recent years, several studies have highlighted the efficacy of DNA barcoding in species identification and biodiversity assessment (Meier et al., 2006). These studies have demonstrated the utility of DNA barcoding across diverse taxa, including crustaceans, and underscore its potential for enhancing our understanding of species diversity and distribution patterns.
In addition to genetic factors, heavy metal contamination in marine crabs is an emerging concern with significant implications for public health. Heavy metals, such as mercury, lead, cadmium, and arsenic, can accumulate in marine organisms, entering the food chain and posing serious health risks to humans who consume contaminated seafood. Long-term exposure to these metals has been linked to various health issues, including neurological disorders, kidney damage, and increased risk of cancers (Doi and Minegishi, 2020; Huo et al., 2016). The concentrations of these contaminants in edible species like P. pelagicus and P. segnis can vary significantly based on environmental factors and geographical location, rendering regular monitoring and risk assessments crucial (Zhou et al., 2022). By integrating genetic and environmental assessments, this study aims to provide comprehensive insights into the health and sustainability of Portunus pelagicus and P. segnis populations, contributing to better fisheries management practices while safeguarding consumer health. Anthropogenic activities such as industrial discharge, mining operations, and agricultural runoff contribute to the release of these metals into marine ecosystems, where they can accumulate in aquatic organisms, posing risks to human health and marine biodiversity. The assessment of heavy metal concentrations in marine organisms is therefore crucial for understanding environmental contamination levels and evaluating potential risks to human consumers (Shi et al., 2019). Additionally, molecular approaches, such as DNA barcoding using partial mitochondrial cytochrome c oxidase subunit I (COI) gene sequences, offer complementary insights into species identification, genetic diversity, and population structure of Portunus spp. (Lai et al., 2010). This study presents a comprehensive assessment of heavy metal concentrations and partial COI gene sequences of Portunus spp., focusing on the blue swimmer crab (P. pelagicus) and closely related congeners. By combining chemical analyses of heavy metal levels with molecular data, we aim to elucidate patterns of metal accumulation, assess potential health risks associated with crab consumption, and enhance our understanding of evolutionary linkages and genetic diversity within the genus Portunus. Through the integration of chemical and molecular approaches, this research aims to provide insightful information about the ecological health of coastal ecosystems, the sustainability of seafood resources, and the genetic diversity of swimming crabs. Such insights are essential for informing management and conservation strategies aimed at preserving marine biodiversity and safeguarding human health. This study represents a significant contribution to the fields of environmental toxicology, molecular ecology, and seafood safety, with implications for both scientific research and policymaking in the realm of marine conservation and public health.
MATERIALS AND METHODS
Collection of samples and morphological analysis
Samples comprising 210 fresh crabs and tin-packed crab meat were collected from the local fisheries market at West Wharf Fish Harbor (24.8536° N, 66.9836° E) and a seafood processing plant at Korangi Fish Harbor (24.8844° N, 67.1443° E). Upon collection, specimens underwent morphological assessment and were promptly stored in ice before transfer to the Aquatic Diagnostics and Research Centre in Karachi, Pakistan. Species identification was conducted up to the possible species level using morphological traits (Jirapunpipat et al., 2008). The first walking leg was carefully removed, sliced, and immediately preserved in 99% ethanol for subsequent DNA analysis. Species were identified based on external morphology, followed by DNA extraction for analyzing the COI gene barcode region in each specimen (Lu et al., 2022).
Extraction of DNA
The genomic DNA extracted from crab muscles was isolated using the Bio-Basic DNA isolation kit BS-88504. The extracted DNA was resuspended in 80 μL of T.E buffer, and its concentration was spectrophotometrically estimated before being stored in the freezer until further use. To eliminate protein contaminants, 2 μL of RNase with 50 μL of TE buffer was added to the sample, followed by incubation in a water bath for 2 h at 37°C. DNA quantification was performed using both gel electrophoresis and the spectrophotometric method (Sambrook and Russell, 2001).
PCR amplification
Dilutions were adjusted to a standard 1:20 ratio to achieve a working concentration of 15 ng DNA/μL. PCR amplification of the partial coding regions of the cytochrome oxidase subunit 1 (COI) gene was conducted using the primer set LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’), as described by Folmer et al. (1994). The PCR reaction mixture (total volume 25 μL) consisted of 0.5 units of GoTaq DNA polymerase (Promega), 1.2 mM MgCl2, 0.3 μM of each primer, 200 μM of each dNTP, and 15 ng of genomic DNA. PCR amplification was carried out using a temperature gradient thermocycler. The PCR conditions comprised an initial denaturation step at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 42°C for 1 min, and extension at 72°C for 2 min. A final elongation step at 72°C for 7 min concluded the PCR program. The amplified products were visualized by electrophoresis in 1x TBE buffer stained with SYBR green on a 1.5% agarose gel at 60 mA. Negative controls were included in each amplification assay, and a 1 kb molecular weight marker (Nippon Genetics) served as a reference for band size.
DNA sequencing and analysis
PCR products from each sample were purified using the FastGene Gel/PCR extraction kit (Biobasic). The purified DNA fragments were then submitted for sequencing to Macrogen Company (Seoul, Korea). DNA sequence data were analyzed using applied biosystems sequence scanner v1.0 software (SPSS, Chicago, IL). Sequence results were utilized for species identification, initially searching for sequence similarity via the NCBI BLAST (basic local alignment search tool) website (www.ncbi.nlm.nih.gov/BLAST). Sequences were aligned using the Clustal W tool in MEGA X (MEGA Inc., Ocheyedan, IA) and submitted to GenBank for accession numbers.
Heavy metal analysis
The carapace meat samples from tin-packed blue swimming crabs were transported in dry ice to the Centralized Science Laboratories at the University of Karachi (UoK). Eight grams (dry weight) of meat samples were precisely weighed and transferred to digestion tubes. To aid the digestion, 5 mL of concentrated nitric acid and 5 mL of concentrated sulfuric acid were applied to the sample. The mixture was heated on a hot plate at 60°C for 30 min or until it approached near dryness. After cooling, additional nitric acid was added up to 10 mL to oxidize any remaining organic matter. The gradual addition of nitric acid continued until effervescence ceased, and the digestion was returned to the hot plate for an additional hour.
Following cooling, a small quantity of deionized water was added to increase the volume to approximately 20 mL. The digests were then filtered using Whatman 41 filter paper into 30 mL volumetric flasks and made up to volume with deionized water.
Metal estimation of aluminum (Al), chromium (Cr), zinc (Zn), copper (Cu), lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg), cobalt (Co), and iron (Fe) in the digests were performed using an Atomic Absorption Spectrometer (AAS) 3100 by Perkin Elmer (USA), model Analyst 700. The analyses were conducted at least in triplicate, and all concentrations are expressed in milligrams of element per hundred grams of fresh mass (mg/100g).
RESULTS AND DISCUSSION
The findings of this study underscore the complex interplay between genetic diversity and environmental factors influencing the health of blue swimmer crab populations. The significant genetic variability found among P. pelagicus populations in China (Jiang et al., 2021) reinforces the notion that maintaining genetic diversity is critical for the resilience of species in the face of environmental changes. Lai et al. (2023) further elucidated how environmental conditions affect genetic structures, suggesting that fisheries management should consider local environmental dynamics to enhance the sustainability of crab populations.
Additionally, the rising concern over heavy metal contamination in marine crabs, as discussed by Doi and Minegishi (2020) and highlighted by Zhou et al. (2022) presents a significant challenge for both ecological health and food safety. The accumulation of metals such as mercury and cadmium in P. pelagicus and P. segnis raises alarms regarding the potential health risks for consumers, necessitating stricter regulatory frameworks and monitoring programs to mitigate these risks and protect public health.
Furthermore, the interplay between genetic health and environmental stressors, including pollution, indicates that effective management strategies must be multi-faceted. By drawing on recent findings regarding genetic assessments and contamination levels, stakeholders can develop targeted conservation efforts that not only preserve genetic diversity but also safeguard the health of marine ecosystems and human populations.
Genetic diversity and haplotype networks
COI mtDNA sequences were aligned using Clustal-X, and their alignment quality was verified through correct amino acid translation. Nucleotide diversity (π) and haplotype diversity (h) were calculated for the entire population and individual sampling locations. The analysis involved 21 nucleotide sequences, resulting in a final dataset of 687 positions. For Portunus segnis, the sequence length was 621 bp with a nucleotide composition of A=25.1%, C=21.3%, G=18.2%, and T=35.4%. For Portunus. pelagicus, the sequence length was 676 bp with a nucleotide composition of A=26.5%, C=20.6%, G=17.5%, and T=35.5%. Pairwise genetic distances among Egypt’s five crab species sampled showed the highest value (0.038) between L. corrugatus and P. pelagicus, and the lowest (0.148) between C. hellerii and C. natator (Table I).
A median-joining haplotypic network was created to illustrate the genetic relationships among the sequenced crab samples and those retrieved from GenBank. The disparity index per site for each sequence pair showed an overall average of 0.022, indicating significant differences in base composition biases, with P-values smaller than 0.05 considered significant. The nucleotide diversity (π) was 0.151. All analyses were conducted using MEGA11.
Table I. Nucleotide frequencies of both crab species in (%).
|
Portunus spp. |
T |
C |
A |
G |
Total |
|
Portunus segnis |
35.4 |
21.3 |
25.1 |
18.2 |
621 |
|
Portunus pelagicus |
35.5 |
20.6 |
26.5 |
17.5 |
676 |
Base substitution analysis
The number of base substitutions per site between sequences is presented, with standard error estimates shown above the diagonal, obtained through a bootstrap procedure with 300 replicates. Analyses were conducted using the Tajima-Nei model, with the rate variation among sites modeled with a gamma distribution (shape parameter=1). The analysis included 21 nucleotide sequences, and all ambiguous positions were removed for each sequence pair using the pairwise deletion option. The final dataset comprised 687 positions. Results from Tajima’s Neutrality Test are detailed in (Table II). The number of base substitutions per site between sequences is shown. Standard error estimates are shown above the diagonal and were obtained through a bootstrap procedure (300 replicates). Analyses were conducted using the Tajima-Nei model (Tajima and Nei, 1984). The rate variation among sites was modeled with a gamma distribution (shape parameter =1). This analysis involved 21 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 687 positions in the final dataset Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021) (Supplemenaty Table I).
Table II. Results from Tajima’s neutrality test.
|
Number of sequences (m) |
21 |
|
Number of segregating sites (S) |
255 |
|
Ps (S/n) |
0.371179 |
|
Θ (Ps/a1) |
0.103170 |
|
Nucleotide diversity (π) |
0.151237 |
|
Tajima test statistic (D) |
1.921004 |
m, number of sequences; n, total number of sites; S, Number of segregating sites; ps, S/n; Θ, ps/a1; π, nucleotide diversity, and D is the Tajima test statistic.
Phylogenetic analysis
A phylogenetic tree was constructed using maximum parsimony for 21 species of Portunus, with sequences from GenBank serving as references. The phylogenetic tree, supported by 250 bootstrap replicates, forms a well-supported clade (Fig. 1). The analysis included P. segnis and P. pelagicus (OL840323 and OL840324) along with sequences
Table III. Essential and toxic trace elements content (mg/100g) in the meat of Portunus pelagicus and Portunus segnis crabs (mg/100g) in non-monsoon and in monsoon BDL Below the Detection Limit < 0.00000001 mg/100g.
|
crabs samples mean values |
Essential trace elements |
Toxic trace elements |
||||||||
|
Iron |
Chromium |
Zinc |
Copper |
Cobalt |
Cadmium |
Mercury |
Arsenic |
Lead |
Aluminum |
|
|
Non-monsoon |
||||||||||
|
Portunus pelagicus |
446.7 |
0.057 |
3.63 |
1.21 |
0.612 |
0.103 |
0.0000000255 |
0.011 |
0.013 |
0.222 |
|
Portunus segnis |
573.2 |
0.015 |
7.61 |
1.16 |
0.904 |
0.024 |
0.0000000314 |
0.065 |
0.139 |
0.0834 |
|
Mean |
509.5 |
0.036 |
5.62 |
1.185 |
0.758 |
0.0635 |
0.0000000284 |
0.038 |
0.076 |
0.0528 |
|
Monsoon |
||||||||||
|
Portunus pelagicus |
328.7 |
0.957 |
2.63 |
1.62 |
0.612 |
0.245 |
BDL |
0.589 |
0.071 |
0.101 |
|
Portunus segnis |
127.8 |
0.715 |
9.61 |
1.87 |
0.304 |
0.098 |
BDL |
2.647 |
0.436 |
0.0971 |
|
Mean |
456.5 |
0.836 |
6.12 |
1.745 |
0.458 |
0.171 |
BDL |
1.618 |
0.253 |
0.086 |
GU321237, MZ393893, MZ393892, MZ393886, OL588010, MW264449, MW277922, KT365746, KT365745, KT365747, KT365742, KT365743, KT365737, KT365736, KT365738, JX398098, JX398099, and JX398092 from GenBank, based on the COI region. Using the maximum likelihood method and the general time reversible model, the tree with the highest log likelihood (-4948.76) was selected. The tree’s branches indicate the percentage of trees in which the associated taxa clustered together. Initial trees for the heuristic search were obtained using Neighbor-Join and BioNJ algorithms applied to a pairwise distance matrix estimated with the maximum composite likelihood (MCL) approach, selecting the topology with the superior log likelihood value. Branch lengths are measured in substitutions per site. The analysis, involving 21 nucleotide sequences and a total of 687 positions, showed two major clades. One clade grouped P. pelagicus, P. segnis, P. armatus and P. reticulatus with all representatives of the family Portunidae. Evolutionary analyses were conducted in MEGA11. The evolutionary history among OL840324, OL840323, KY587391, KY587767, KY695086, KY587390, KY695087, KY587389, KY587772, KY587387, MF002106, and KY587386 was concluded by using the Maximum Likelihood method and the Kimura 2-parameter model. The bootstrap consensus tree, constructed from 100 replicates, represents the evolutionary history of the taxa analyzed, with branches having less than 50% of bootstrap support being collapsed. The final dataset comprised 768 positions, and evolutionary analyses were conducted in MEGA11 (Fig. 2).
Trace elements
The study found that various factors, including season, length, weight, and the physical and chemical status of water, influence metal accumulation in tissues. (Zaynab et al., 2022). The mean carapace width (CW), mean carapace length (CL), and weights of the species examined were similar for P. segnis and P. pelagicus (p > 0.05). During the monsoon season, higher concentrations of copper, lead, cadmium, and aluminum were detected, followed by iron, zinc, copper, cobalt, chromium, and mercury. The essential trace elements were ordered as Fe > Zn > Cu > Co > Cr, while the toxic trace elements were ordered as Pb>Cd>Al>As > Hg (Table III).
In the non-monsoon season, higher amounts of arsenic, lead, cadmium, and chromium were detected compared to the monsoon season. The essential trace elements were ordered as Fe>Zn>Cu>Cr>Co and the toxic trace elements were ordered as As>Pb>Cd>Al>Hg (Table III). The blue swimming crab accumulates heavy metals such as iron, zinc, lead, and cadmium throughout the year, with significant increases in arsenic during the non-monsoon season and lead during the monsoon season. These concentrations were higher than the permissible limits recommended by FAO (1998), posing health risks.
The accumulation of trace elements is influenced by factors such as salinity, pH, metal bioavailability, tissue composition, pollution load, and environmental hydrodynamics. Essential trace elements like iron, copper, zinc, cobalt, and chromium are vital for human physiological and biochemical processes, and crab meat is a good source of these minerals. However, values varied significantly among crab species due to factors like seasonal variation, species, age, sex, environmental conditions, and feeding patterns. Previous studies found that copper concentrations were higher than lead, cadmium, and chromium in P. pelagicus and P. segnis muscle tissues during the monsoon season. The study concluded that both P. pelagicus and Portunus segnis tend to accumulate heavy metals, with increased industrialization leading to marine pollution, disrupting the food chain, and adversely affecting human health.
The genetic diversity and evolutionary relationships of the blue swimming crabs, Portunus segnis and Portunus pelagicus, were analyzed using COI mtDNA sequences. P. segnis exhibited a sequence length of 621 bp with nucleotide frequencies of A=25.1%, C=21.3%, G=18.2%, and T=35.4%, while P. pelagicus had a sequence length of 676 bp with nucleotide frequencies of A=26.5%, C=20.6%, G=17.5%, and T=35.5%. The genetic distances between the crab species sampled in Egypt showed the highest value (0.038) between L. corrugatus and P. pelagicus, and the lowest (0.148) between C. hellerii and C. natator. Median-joining haplotype networks (MJN) visualized the evolutionary connections among the crab samples, revealing moderate genetic variation within the populations (π = 0.151) (Tamura et al., 2021). Disparity Index analysis indicated significant base composition biases among the sequences (P < 0.05). The significant genetic variability found among Portunus populations aligns with the work of Akin and Ozturk (2023) utilized COI gene sequences for molecular identification and phylogenetic characterization of this species. Their research underscores the utility of molecular markers in delineating species and understanding evolutionary relationships within the Portunidae family, which is essential for informed conservation strategies.
Phylogenetic relationships among 21 Portunus species were inferred using maximum likelihood and maximum parsimony methods. The phylogenetic tree constructed from COI sequences revealed two major clades within the Portunidae family, with strong bootstrap support (250 replicates). This analysis incorporated both newly obtained sequences and reference sequences from GenBank, demonstrating clear genetic distinctions among P. pelagicus, P. segnis, P. armatus and P. reticulatus (Tamura et al., 2021; Kimura, 1980). Trace element analysis of P. segnis and P. pelagicus showed that these species accumulate various metals in their tissues. Higher concentrations of iron, zinc, copper, chromium, and cobalt as essential and toxic trace elements arsenic, lead, cadmium, and aluminum were detected during the non-monsoon season.
While Fe>Zn>Cu>Co>Cr are essential trace elements and lead, cadmium, aluminum, and arsenic were more prevalent during the monsoon season as toxic elements. Seasonal variations significantly affected arsenic, lead, cadmium, aluminum, and cobalt concentrations. However, the levels of these elements exceeded FAO (1998) permissible limits, posing potential health risks. Environmental factors such as salinity, pH, metal bioavailability, and pollution influence trace element accumulation in crabs. Industrialization and marine pollution contribute to heavy metal bioaccumulation in marine organisms, disrupting the food chain and impacting human health (Blewett and Wood, 2015). P. pelagicus and P. segnis demonstrated a considerable capacity to accumulate heavy metals, indicating the need for regular monitoring to mitigate health risks associated with their consumption (Monastero et al., 2017). These findings underscore the importance of understanding genetic diversity, evolutionary relationships, and environmental impacts on marine species to ensure sustainable management and public health safety.
CONCLUSIONS
Our study investigates the genetic diversity, phylogeography, and trace metal accumulation in Portunus pelagicus and Portunus segnis crabs in the Northern Arabian Sea. Using the cytochrome oxidase subunit 1 (COI) gene for DNA barcoding, we found significant haplotype diversity and low nucleotide diversity, suggesting potential cryptic diversity. Population expansion within the Arabian Sea was indicated by neutrality tests and mismatch analysis, showing dynamic evolutionary processes. Phylogenetic analyses revealed significant phylogeographic structuring among Portunus spp., highlighting the importance of considering spatial genetic variation in conservation and management. Combining genetic and morphological data allowed for accurate species identification despite morphological variability. We also found seasonal fluctuations in heavy metal concentrations in crab tissues, with levels exceeding FAO (1998) limits, posing health risks. Our research emphasizes the need for environmental monitoring and regulatory measures to mitigate pollution. This study enhances our understanding of crab populations’ ecological dynamics and genetic diversity in the Northern Arabian Sea, supporting integrated approaches in molecular genetics, environmental science, and fisheries management to preserve marine biodiversity and protect human health.
Declarations
Funding
The study received no external funding.
There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20240712071015
Statement of conflict of interest
The authors have declared no conflict of interest.
REFERENCES
Akin, A.T. and Ozturk, S.B., 2023. Molecular identification and phylogenetic characterization of the blue swimming crab (Portunus pelagicus) using COI gene sequences. J. Mar. Sci. Eng., 11: 867.
Blewett, T.A. and Wood, C.M., 2015. Low salinity enhances NI-mediated oxidative stress and sub-lethal toxicity to the green shore crab (Carcinus maenas). Ecotoxicol. environ. Safe., 122:159–170. https://doi.org/10.1016/j.ecoenv.2015.07.019
Doi, H. and Minegishi, Y., 2020. Heavy metal contamination in marine crabs: A review of the ecotoxicological impact and human health risk. Environ. Int., 144: 106025.
Food and Agriculture Organization (FAO), 2016. The State of world fisheries and aquaculture 2016: Contributing to food security and nutrition for all. Rome, pp. 200.
FAO, 1998. Carbohydrates in human nutrition. Report on a Joint FAO/ WHO Expert Consultation. FAO Food and Nutrition Paper No. 66 Food and Agriculture Organization of the United Nations: Rome.
Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech., 3: 294–299.
Huo, S., Luo, H., Kong, T., Wang, Z. and Liu, X., 2016. Heavy metal concentrations in marine crabs from coastal areas of northern Beibu Gulf, South China Sea. Environ. Monit. Assess., 188: 1-12.
Jiang, D., Wu, M., Geng, J., Xu, H. and Yin, Y., 2021. Genetic diversity and population structure of the blue swimming crab (Portunus pelagicus) in China. Sci. Rep., 11: 1-12.
Jirapunpipat, K., Aungtonya, C. and Watanabe, S., 2008. Morphological study and application of multivariate analysis for the mud crab genus Scylla in Klongngao mangrove, Ranong province, Thailand. Phuket Mar. Biol. Center Res. Bull., 69: 7–24.
Joesidawati, M.I., Nursalim, N., Kholilah, N., Kurniasih, E.M., Cahyani, N.K.D. and Ambariyanto, A., 2023. Utilizing DNA barcoding approach to study the diversity of the blue swimming crab from Tuban District, East Java, Indonesia. Biodiversitas, 24: 4731–4737. https://doi.org/10.13057/biodiv/d240913
Kazmi, Q.B., 2003. Taxonomic studies of crustaceans in Pakistan. In: Global taxonomy institute in Asia (ed. J. Shimura), Report and Proc. 1st GIT Regional Workshop in Asia Putrajaya, Malaysia. pp. 230-248.
Kimura, M., 1980. A simple method for estimating the evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. mol. Evol., 16: 111-120. https://doi.org/10.1007/BF01731581
Lai, J.C.Y., O’Meally, D. and Lee, S.Y., 2023. Environmental influence on the genetic structure of blue swimming crabs (Portunus pelagicus): Implications for fisheries management. J. exp. Mar. Biol. Ecol., 556: 151827.
Lai, J.C.Y., Ng, P.K.L. and Davie, P.J.F., 2010. A revision of the Portunus pelagicus (Linnaeus, 1758) species complex (Crustacea: Brachyura: Portunidae), with the recognition of four species. Raffles Bull. Zool., 58: 199-237.
Lu, Y.M., Shih, C.H., Chen, P.C., Kao, W.C., Lee, Y.C., Han, Y.S. and Tzeng, T.D., 2022. Genetic variations and expansion of the blue swimmer crab (Portunus pelagicus) in Southeast Asia. J. Mar. Sci. Eng., 10: 1071. https://doi.org/10.3390/jmse10081071
Maryani, L., Bengen, D.G. and Nurjaya, I.W., 2023. Distribution and growth patterns of crab (P. pelagicus) based on environmental characteristics in Candi Waters, Pamekasan Regency, East Java Province. J. Kelautan Trop., 26: 340–348. https://doi.org/10.14710/jkt.v26i2.17322
Meier, R., Shiyang, K., Vaidya, G. and Ng, P.K., 2006. DNA barcoding and taxonomy in Diptera: A tale of high intraspecific variability and low identification success. Syst. Biol., 57: 1-17.
Monastero, R.N., Karimi, R., Nyland, J.F., Harrington, J., Levine, K. and Meliker, J.R., 2017. Mercury exposure, serum antinuclear antibodies, and serum cytokine levels in the Long Island Study of Seafood Consumption: A cross-sectional study in NY, USA. Environmental Research, pp. 334–340. https://doi.org/10.1016/J.ENVRES.2017.03.037
Noori, A., Moghaddam, P., Kamrani, E., Akbarzadeh, A., Neitali, B.K. and Pinheiro, M.A.A., 2015. Condition factor and carapace width versus wet weight relationship in the blue swimming crab Portunus segnis. Anim. Biol., 65: 87. https://doi.org/10.1163/15707563-00002463
Saher, N.U., Naz, F. and Kamal, M., 2019. Mitochondrial DNA variation and population genetic structure of mud crab, Scylla serrata from Pakistan/Northern Arabian Sea. Genet. Aquat. Organ., 3: 67–77. https://doi.org/10.4194/2459-1831-v3_2_04
Sambrook, J. and Russell, D.W., 2001. Molecular cloning: A laboratory manual, 3rd ed., Vol. 1. Cold Spring Harbor Laboratory Press.
Shi, L., Liu, G., Zhang, M., Yao, L. and Li, X., 2019. Distribution and source identification of heavy metals in surface sediments of the East China Sea. Mar. Pollut. Bull., 146: 436-445.
Siddiqui, P.J.A., Farooq, S., Shafique, S., Burhan, Z.N. and Farooqi, Z., 2008. Conservation and management of biodiversity in Pakistan through the establishment of marine protected areas. Ocean Coast. Manage., 51: 377–382. https://doi.org/10.1016/j.ocecoaman.2008.01.006
Tajima, F. and Nei, M., 1984. Estimation of evolutionary distance between nucleotide sequences. Mol. Biol. Evol., 1: 269-285.
Tamura, K., Stecher, G. and Kumar, S., 2021. MEGA 11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol., 38: 3022-3027. https://doi.org/10.1093/molbev/msab120
Zaynab, M., Al-Yahyai, R., Ameen, A., Sharif, Y., Ali, L., Fatima, M., Khan, K.A. and Li, S., 2022. Health and environmental effects of heavy metals. J. King Saud Univ. Sci., 34: 101653. https://doi.org/10.1016/j.jksus.2021.101653
Zhou, Y., Zheng, H., Chen, R. and Wu, F., 2022. Trace metal accumulation in blue swimming crabs (Portunus pelagicus) from the South China Sea: Influencing factors and health risk assessment. Mar. Pollut. Bull., 175: 113394.