Portunus pelagicus (Linnaeus, 1758) as a Sentinel Species to Assess Trace Metal Occurrence: A Case Study of Kuwait Waters (Northwestern Arabian Gulf).

Heavy metal pollution can adversely impact marine life, such as crabs, which can accumulate it in different organs and potentially transfer and biomagnify along the food chain in aquatic ecosystems. This study aimed to examine the concentrations of heavy metals (Cd, Cu, Pb, and Zn) in sediment, water, and crab tissues (gills, hepatopancreas, and carapace) of the blue swimmer crab Portunus pelagicus in the coastal areas of Kuwait, northwestern Arabian Gulf. Samples were collected from Shuwaikh Port, Shuaiba Port, and Al-Khiran areas. The accumulation of metals in crabs were higher in the carapace > gill > digestive gland, and the highest metal concentration was found in crabs collected from Shuwaikh > Shuaiba > Al-Khiran. The metal concentrations in the sediments were in the order Zn > Cu > Pb > Cd. Zn was the highest metal concentration detected in marine water sampled from the Al-Khiran Area, whereas the lowest metal was Cd sampled in water from the Shuwaikh Area. The results of this study validate the marine crab P. pelagicus as a relevant sentinel and prospective bioindicator for evaluating heavy metal pollution in marine ecosystems.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression.

BACKGROUND: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. RESULTS: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four ß sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. CONCLUSION: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

The first description of the blue swimming crab (Portunus pelagicus) transcriptome and immunological defense mechanism in response to white spot syndrome virus (WSSV).

In the global shellfish farming industry, white spot syndrome virus (WSSV) is a major cause of mortality and a significant factor in economic losses. However, information on molecular immune responses to WSSV in blue swimming crabs (Portunus pelagicus) has never been reported. First, viral loads were measured in the gills, hepatopancreas, intestines, subcuticular epithelium and hemocytes of blue swimming crabs (50 ± 10 g) (n = 4) after WSSV induction at 0, 24, 48 and 96 h post injection (hpi). A significant increase in WSSV particles was observed in gills at 48 and 96 hpi, as supported by histopathology. To further investigate the acute immune response to WSSV, total RNA from the same gill tissues at 0, 24, and 96 hpi was used to construct 16 high-quality RNA-seq cDNA libraries. In summary, 162,740 unigenes were discovered in these transcriptomic libraries analyzed with the GO, KO, KOG, NR, NT, PFAM and SwissProt databases. Intensive sequence analysis against control crabs using three major categories of gene oncology (GO) of DEGs, biological processes (BPs), molecular functions (MFs), and cellular components (CCs), indicated that induction of WSSV in blue swimming crabs strongly affected the immune responses of the target animals significantly during the early stages of infection from 24 to 96 hpi. Furthermore, KEGG identified approximately twenty biological pathways of gene expression that were both downregulated and upregulated. Interestingly, at 24 and 96 hpi, several immune-related genes involved in virus defense in the blue swimming crab, particularly crustin 2, chitinase, anti-lipopolysaccharide, proteinase inhibitor, and lysozyme, were highly expressed during the WSSV early infection stages. At the same time, viral mRNA transcripts, including WSV289, WSV343, WSV306, deoxyuridine 5' triphosphate nucleohydrolase, RING finger containing E3 ubiquitin-protein ligase WSV403 and WSV404, were recorded in the top twenty upregulated genes. Moreover, some immune-responsive genes related to growth development, such as chitinase, tubulin alpha and beta chains, trypsin, and the cathepsin family, were also differentially expressed during these periods. Expression validation of 20 upregulated and 11 downregulated immune-related genes using qRT‒PCR showed similar patterns with transcriptome information. Overall, the data showed that during WSSV infection, a number of immune-, metabolism-, and growth-related pathways were activated, and several of the pathways involved differed depending on the stage of virus invasion. These findings could effectively help us better understand the impact of WSSV on the physiology of blue swimming crabs and serve as a valuable reference for future research on the immune system and disease control in this target species.

The effect of scaffold hydroxyapatite derived from portunus pelagicus shell on the expression of fibroblast growth factor-2 (fgf-2) and bone morphogenetic proteins-2 (bmp -2) in the extraction sockets of cavia cobaya / El efecto del scaffold de hidroxiapatita derivada de portunus pelagicus sobre la expresión del factor de crecimiento de fibroblastos-2 (FGF-2) y las proteínas morfogenéticas óseas-2 (BMP-2) en los alvéolos dentarios de cavia cobaya

Objetive: To determine the expression of Fibroblast Growth Factor (FGF)-2 and Bone Morphogenetic Protein (BMP)-2 after application of scaffold hydroxyapatite from Rajungan crab shell (Portunus pelagicus) in the tooth extraction socket of Cavia cobaya. Material and Methods: This study used a post-test only control group design with 28 Cavia cobaya separated into two groups, control and treatment group. The left mandibular incisor was extracted, and socket preservation was conducted. A hydroxyapatite graft derived from crab shells was mixed with gelatin and eventually turned into a scaffold, which was afterward put into the extraction socket. After 7 days and 14 days, each group was terminated and examined using immunohistochemical staining to observe the expression of FGF-2 and BMP-2. One-Way Anova and Tukey HSD were used to examine the research data. Results: FGF-2 and BMP-2 expressions were observed higher in the group that received hydroxyapatite scaffold at the post-extraction socket than those in the group that did not receive hydroxyapatite scaffold. Conclusion: The application of a hydroxyapatite scaffold from Rajungan crab shell (Portunus pelagicus) to the tooth extraction socket can increase FGF-2 and BMP-2 expression.

Objetivo: Determinar la expresión del factor de crecimiento de fibroblastos (FGF)-2 y la proteína morfogenética ósea (BMP)-2 después de la aplicación de hidroxiapatita de andamio de caparazón de cangrejo Rajungan (Portunus pelagicus) en el alvéolo de extracción dental de Cavia cobaya. Material y Métodos: Este estudio utilizó un diseño de grupo de control solo posterior a la prueba con 28 Cavia cobaya separados en dos grupos, grupo de control y grupo de tratamiento. Se extrajo el incisivo mandibular izquierdo y se realizó la preservación del alvéolo. Un injerto de hidroxiapatita derivado de caparazones de cangrejo se mezcló con gelatina y se convirtió en un andamio, que luego se colocó en el alvéolo de extracción. Después de 7 días y 14 días, se terminó cada grupo y se examinó mediante tinción inmunohistoquímica para observar la expresión de FGF-2 y BMP-2. Se utilizaron One-Way Anova y Tukey HSD para examinar los datos de la investigación. Resultados: Las expresiones de FGF-2 y BMP-2 se observaron más altas en el grupo que recibió la estructura de hidroxiapatita en el alvéolo posterior a la extracción que en el grupo que no recibió la estructura de hidroxiapatita. Conclusión: La aplicación de un andamio de hidroxiapatita de caparazón de cangrejo Rajungan (Portunus pelagicus) al alvéolo de extracción dental puede aumentar la expresión de FGF-2 y BMP-2.

Reproductive Biology and Feeding Ecology of The Blue Swimming Crab (Portunus pelagicus) in Northern Coastal Waters, Sri Lanka.

Blue swimming crab (Portunus pelagicus) fishery has emerged to become an important export-oriented fishery in Sri Lanka over a decade and recently resulted in a rapid increase in the exploitation. The present study attempts to understand the reproductive biology and feeding ecology of blue swimming crab which will be vital in the management of capture fishery. Five major landing sites in the Jaffna district in Northern Sri Lanka, where blue swimming crab is frequently landed throughout the year were selected for the study. Biological parameters relating to sex, carapace width, body weight, maturity, and egg sac colour with egg sac weight were recorded at the field from November 2014 to October 2015. Randomly selected crab samples were brought to the laboratory and analysed for their maturity stages, length at first maturity, Gonadosomatic Index, fecundity, and gut contents. The study revealed that male crabs mature at a smaller size than females. The sex ratio varied greatly with time and males were always dominant in the catch. The blue swimming crabs in the Northern waters of Sri Lanka spawn throughout the year, with two spawning peaks in February and May. The total fecundity of ovigerous blue swimming crab increased with increased carapace width and body weight and it ranged from 123,482 to 3,179,928 eggs, with an average of 884,982 ± 676,420. A remarkably higher percentage of empty stomachs were observed under the present study in both mature and immature crabs and this could be due to lack of food availability in the environment and different digestibility rates of food items. The diet of blue swimming crab is highly variable reflecting the ability to adopt to different modes of feeding.

Comparative population genetics of swimming crab host (Portunus pelagicus) and common symbiotic barnacle (Octolasmis angulata) in Vietnam.

BACKGROUND: By comparing spatial geographical structures of host populations with that of their symbionts light can be shed on their biological interactions, and the degree of congruence between host and symbiont phylogeographies should reflect their life histories and especially dispersal mechanisms. METHODS: Here, we analyzed the genetic diversity and structure of a host, the blue swimming crab, Portunus pelagicus, and its symbiotic pedunculate barnacle Octolasmis angulata from six location sites representing three geographic regions (north, central and south) along the Vietnam coastline. High levels of congruence in their phylogeographic patterns were expected as they both undergo planktonic larval stages. RESULTS: Based on the COI mtDNA markers, O. angulata populations showed higher genetic diversity in comparison with their host P. pelagicus (number of haplotype/individuals, haplotype and nucleotide diversity are 119/192, 0.991 ± 0.002 and 0.02; and 89/160, 0.913 ± 0.02 and 0.015, respectively). Pairwise Fst and AMOVA analyses showed a more pronounced population structure in the symbiotic barnacle than in its crab host. The DAPC analyses identified three genetic clusters. However, both haplotype networks and scatter plots supported connectivity of the host and the symbiotic barnacle throughout their distribution range, except for low subdivision of southern population. Isolation by distance were detected only for the symbiont O. angulata (R2 = 0.332, P = 0.05), while dbMEM supported spatial structure of both partners, but only at MEM-1 (Obs. 0.2686, P < 0.01 and Obs. 0.2096, P < 0.01, respectively).

Data on population density, growth, survival, water quality, larval stage index and ingestion rate of selected microalgae of portunids crabs at different feeding regimes.

Population density, growth, survival, water quality and larval stage index of purple mud crab, Scylla tranquebarica at different feeding regimes and the data on ingestion rate of chosen microalgae, survival and larval development of blue swimming crab, Portunus pelagicus are presented. A twenty days of S. tranquebarica larval culture from zoeal 1 until megalopa stage under two different feeding regimes of A) Rotifer, Artemia nauplii and shrimp meat and B) Rotifer, Artemia nauplii and artificial feed is shared. A method on investigation of individual larvae of P. pelagicus capability to catch four different types of microalgae within 24 h is also shared. Direct eye observation, data collected through the larval rearing culture of S. tranquebarica and further statistical analysis were done daily until the crabs reached the megalopa stage. The result obtained from the optimum density of selected microalgae fed by individual larvae of P. pelagicus will be combined with the highest survival rate and larval stage index to develop feeding schedule for crab larvae P. pelagicus. This dataset has not previously been published and is of great potential for further comparison with other - and future investigation of various feeding regimes affected the crab culture. The collected information could be used as a standard feeding regime for nursery and hatchery seed production of others portunids crabs. The data described in this article are available as a supplementary file to this article.

A Glycosaminoglycan Extract from Portunus pelagicus Inhibits BACE1, the ß Secretase Implicated in Alzheimer's Disease.

Therapeutic options for Alzheimer's disease, the most common form of dementia, are currently restricted to palliative treatments. The glycosaminoglycan heparin, widely used as a clinical anticoagulant, has previously been shown to inhibit the Alzheimer's disease-relevant ß-secretase 1 (BACE1). Despite this, the deployment of pharmaceutical heparin for the treatment of Alzheimer's disease is largely precluded by its potent anticoagulant activity. Furthermore, ongoing concerns regarding the use of mammalian-sourced heparins, primarily due to prion diseases and religious beliefs hinder the deployment of alternative heparin-based therapeutics. A marine-derived, heparan sulphate-containing glycosaminoglycan extract, isolated from the crab Portunus pelagicus, was identified to inhibit human BACE1 with comparable bioactivity to that of mammalian heparin (IC50 = 1.85 µg mL-1 (R2 = 0.94) and 2.43 µg mL-1 (R2 = 0.93), respectively), while possessing highly attenuated anticoagulant activities. The results from several structural techniques suggest that the interactions between BACE1 and the extract from P. pelagicus are complex and distinct from those of heparin.

Evaluation of Formulated Diets Enriched by Spinach Extracts for the Broodstock Females, Portunus pelagicus (Linnaeus, 1758).

BACKGROUND AND OBJECTIVE: Spinach extract (Amaranthacea tricolor) is a very prospective feed raw materials to stimulate the molting and growth of crab female broodstock. The purpose of this study was to evaluate the quality of broodstock females, Portunus pelagicus (Linnaeus, 1758) at different levels of spinach extract based on physical, organoleptic and chemical tests. MATERIALS AND METHODS: Four different treatments of spinach extract (0, 250, 500 and 750 ng g-1 crab, respectively) were used in this study. The female crab samples were collected from coastal region of Padang, West Sumatera and placed randomly in four concrete tanks (200×100×100 cm). Each concrete tank consist of five units of plastic box (45.5×32.5×16.5 cm) with the maximum density was one crab per box. RESULTS: The organoleptic test showed that formulated diets enriched with spinach extract (0, 250 and 500 ng g-1 crab) had a smooth texture, pungent aroma and brown. Whereas from the physics test results obtained good water stability (rupture velocity ranged from 89.20-105.40 min and solids dispersion ranged from 4.97-7.17%), hardness (92.66-98.07%) and sinking velocity (3.88-5.88 cm sec-1) (p<0.05). The results also showed that formulated diet enriched with spinach extract doses of 250-750 ng g-1 crab gave a value of delicacy of feed (0.195-0.386 g crab-1 weight/day) which was significantly different (p<0.05) with 0 ng g-1 crab (0.445 g crab-1 weight/day). The chemical test shows the moisture content were 11.60%, ash 9.31%, protein 44.38%, fat 7.64% and carbohydrate 14.46%. CONCLUSION: It was concluded that there was a linear relationship between dose of spinach extracts in formulated diet and rupture velocity and a quadratic relationship between dose of spinach extracts in formulated diet and solid disperse, hardness and sinking velocity and delicacy of feed.

De novo assembly of genome and development of polymorphic microsatellite loci in the blue swimming crab (Portunus pelagicus) using RAD approach.

The blue swimming crab (Portunus pelagicus) is a valuable marine fishery resource in Indo-West Pacific Ocean. So far, rare genetic resource of this species is available. In this report, the restriction-site associated DNA (RAD) approach was employed to mine the genomic information and identify molecular markers in P. pelagicus. A total of 0.82 Gbp clean data were generated from the genome of individual "X2A". De novo assembly produced 85,796 contigs with an average length of 339 bp. A total of 45,464 putative SNPs and 17,983 microsatellite loci were identified from the genomes of ten individuals. Furthermore, 31 pairs of primers were successfully designed, with 16 of them exhibiting polymorphism in a wild population. For these polymorphic loci, the expected and observed alleles per locus ranged from 1.064 to 7.314 and from 2 to 11, respectively. The expected and observed heterozygosity per locus ranged from 0.0615 to 0.819 and from 0.0626 to 1.000, respectively. Nine loci showed high informative with polymorphism information content (PIC) > 0.5. Five loci significantly deviated from Hardy-Weinberg equilibrium in the samples analyzed. No linkage disequilibrium was found among the 16 polymorphic microsatellite loci. This study provided massive genetic resource and polymorphic molecular markers that should be helpful for studies on conservation genetics, population dynamics and genetic diversity of P. pelagicus and related crab species.

Searching for crab-borne antimicrobial peptides: Crustin from Portunus pelagicus triggers biofilm inhibition and immune responses of Artemia salina against GFP tagged Vibrio parahaemolyticus Dahv2.

Marine organisms represent a huge source of novel compounds for the development of effective antimicrobial drugs. The present study focus on the purification of the antimicrobial peptide crustin from the haemolymph of the blue swimmer crab, Portunus pelagicus, by blue Sepharose CL-6B matrix assisted affinity column chromatography. Crustin showed a single band with a molecular mass of 17 kDa in SDS-PAGE analysis. The XRD analysis exhibited peaks at 32° and 45° while a distinct peak with a retention time of 1.8 min resulted in high performance liquid chromatography (HPLC) pointing out the crystalline nature and purity of crustin, respectively. Crustin purified from P. pelagicus (Pp-Cru) showed immunological activities, triggering encapsulation, phagocytosis on Sepharose beads and yeast (Saccharomyces cerevisiae) respectively. Furthermore, encapsulation of GFP tagged V. parahaemolyticus in Artemia salina and challenging study were assessed under CLSM and the potential of Pp-Cru was examined in vivo. In addition, the growth reduction and biofilm inhibition potential of Pp-Cru on Staphylococcus aureus, Enterococcus faecalis (Gram- positive bacteria) and Pseudomonas aeruginosa, Escherichia coli (Gram-negative bacteria) was evidenced by inverted and confocal laser scanning microscopic analysis, revealing that 100 µg/ml of Pp-Cru can disrupt the biofilm matrix thereby the thickness of biofilm was significantly reduced. Overall, the present investigation might provide a sensitive platform to realize the significant function of Pp-Cru in crustacean immune mechanism as well as its potential to bacterial growth inhibitor. The functional properties of purified Pp-Cru antimicrobial peptide may lead to a superior understanding of innate immune response in P. pelagicus species, which suggest the promising application for drug development in aquaculture.

Effect of ß-1, 3 glucan binding protein based zinc oxide nanoparticles supplemented diet on immune response and disease resistance in Oreochromis mossambicus against Aeromonas hydrophila.

Recently, several immunostimulants such as ß-glucan, microbial and plant products have been used as dietary supplements to combat disease outbreaks in aquaculture. The present study investigates the potential of Portunus pelagicus ß-1, 3 glucan binding protein based zinc oxide nanoparticles (Ppß-GBP-ZnO NPs) supplemented diet on growth, immune response and disease resistance in Mozambique tilapia, Oreochromis mossambicus. The immune-related protein ß-GBP was purified from the haemolymph of P. pelagicus using Sephadex G-100 affinity column chromatography. Ppß-GBP-ZnO NPs was physico- chemically characterized and experimental feed was formulated. Fish were separately fed with commercial diet (control-group I) and Ppß-GBP (group II, III, IV), Ppß-GBP-ZnO NPs (group V, VI, VII), chem-ZnO NPs (VIII, IX, X) mixed diet at the concentration of 0.001%, 0.002% and 0.004% respectively. Triplicate groups of O. mossambicus were fed with experimental diets twice a day for 30 days. Fish receiving Ppß-GBP-ZnO NPs supplemented diet showed a significant increase (P < 0.05) in growth performance. Cellular immune responses (myeloperoxidase activity, lysozyme activity and reactive oxygen species activity) and humoral immune responses (complement activity, antiprotease activity and alkaline phosphatase activity) were evaluated at an interval of 15 days during the feeding trial. Results demonstrate that both cellular and humoral immune responses were substantially increased (P < 0.05) in fish fed with 0.004% of Ppß-GBP-ZnO NPs supplemented diet than others. Antibiofilm potential of Ppß-GBP-ZnO NPs against Aeromonas hydrophila was visualized through confocal laser scanning microscopy (CLSM), which reveals reduction in the preformed biofilm thickness to 10 µm  at the concentration of 50 µg/ml. Furthermore, after 30 days of feeding trial, fish were challenged with aquatic fish pathogen A. hydrophila (1 × 107 cells ml-1) through intraperitoneal injection. Challenge study displayed a reduced mortality rate in fish fed with diet containing Ppß-GBP-ZnO NPs. Thus our study suggests that dietary supplementation of Ppß-GBP-ZnO NPs at 0.004% may have a potential effect to enhance the immune system and survival of O. mossambicus.

Identification, characterization and immune response of prophenoloxidase from the blue swimmer crab Portunus pelagicus and its antibiofilm activity.

Prophenoloxidase is a conserved Cu-containing enzyme acting as a major defense molecule in the immune response of crustaceans. In the present research, we purified prophenoloxidase from the haemolymph of Portunus pelagicus (Pp-proPO) by Blue Sepharose CL-6B chromatography. Pp-proPO exhibited only one band with molecular weight of 75kDa on SDS-PAGE. The purified Pp-proPO was characterized through X-ray diffraction (XRD) and high-performance liquid chromatography (HPLC). Pp-proPO showed phagocytic activity on the yeast Saccharomyces cerevisiae as well as encapsulation on sepharose CL-6B beads associated with CM sepharose and beads of sodium alginate. Pp-proPO also led to strong agglutination on human erythrocytes. Furthermore, Pp-proPO showed magnified PO activity when altered with activated particles acting as pathogen combined molecular patterns (PAMPs), metal ions or other chemicals. Pp-proPO showed relevant antibiofilm activity on Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. Overall, the above results allowed us to claim that Pp-proPO play a key role in immune defense mechanisms of P. pelagicus crabs, in particular towards microbial pathogens; notably we added basic information to the functional characterization of Pp-proPO, as well as to understand its immunological role in crustaceans defense systems.

Molecular cloning and distribution of oxytocin/vasopressin-like mRNA in the blue swimming crab, Portunus pelagicus, and its inhibitory effect on ovarian steroid release.

This study was aimed to characterize the full length of mRNA of oxytocin/vasopressin (OT/VP)-like mRNA in female Portunus pelagicus (PpelOT/VP-like mRNA) using a partial PpelOT/VP-like sequence obtained previously in our transcriptome analysis (Saetan, 2014) to construct the primers. The PpelOT/VP-like mRNA was 626 bp long and it encoded the preprohormones containing 158 amino acids. This preprohormone consisted of a signal peptide, an active nonapeptide (CFITNCPPG) followed by the dibasic cleavage site (GKR), and the neurophysin domain. Sequence alignment of the PpelOT/VP-like peptide with those of other animals revealed strong molecular conservation. Phylogenetic analysis of encoded proteins revealed that the PpelOT/VP-like peptide was clustered within the group of crustacean OT/VP-like peptide. Analysis by RT-PCR revealed the expression of mRNA transcripts in the eyestalk, brain, ventral nerve cord (VNC), ovary, intestine and gill. The in situ hybridization demonstrated the cellular localizations of the transcripts in the central nervous system (CNS) and ovary tissues. In the eyestalk, the mRNA expression was observed in the neuronal clusters 1-5 but not in the sinus gland complex. In the brain and the VNC, the transcripts were detected in all neuronal clusters but not in the glial cell. In the ovary, the transcripts were found in all stages of oocytes (Oc1, Oc2, Oc3, and Oc4). In addition, synthetic PpelOT/VP-like peptide could inhibit steroid release from the ovary. The knowledge gained from this study will provide more understanding on neuro-endocrinological controls in this crab species.

Genetic structure and historical demography of the blue swimming crab (Portunus pelagicus) from southeastern sea of China based on mitochondrial COI gene.

In this study, the population genetic structure and historical demography of the blue swimming crab, Portunus pelagicus, from southeastern sea of China were investigated using cytochrome c oxidase subunit I (COI) gene of mitochondrion. A total of 889 bp segment of COI gene was sequenced, which showed a high haplotype diversity (0.6833-0.8142) and low nucleotide diversity (0.0021-0.0034). Among 30 haplotypes defined in this study, one (H1) was the most dominant (47.7%) and shared by each locality, while the majority (23) were rare and only existed in one individual. The AMOVA analysis revealed a limited population genetic structure, which suggested a high level of gene flow along the distribution areas of China. This conclusion was supported by the pairwise FST comparison values. The topology of the neighbour-joining tree constructed using 30 haplotypes from four localities presented two distinct clades (clade A and clade B). Meanwhile, three sequences of P. pelagicus downloaded from NCBI database showed a high-level divergence with the individuals collected in our study, which might form a new cryptical species. The individuals of clade B were cryptically embedded in the whole population, with a low frequency (7.7-24.2%), while clade A accounted for 75.8-92.3%. Neutrality tests and mismatch analyses suggested a late Pleistocene population expansion for both clade A (47,000-66,000 years ago) and clade B (74,000-100,000 years ago). This study should provide insight into phylogeny, population genetic structure, conservation genetics, and sustainable management of P. pelagicus.

Changes on biogenic, volatile amines and microbial quality of the blue swimmer crab (Portunus pelagicus) muscle during storage.

Biogenic amines (BAs) are a group of substances with low molecular weight organic compounds such as aliphatic, aromatic or heterocyclic structures that are naturally present in animal tissues. The aim of this study was to investigate the changes on the formation of biogenic amine, bacterial load and biochemical characteristics in blue swimmer crab (Portunus pelagicus) at different storage temperatures (4 and 20 °C) up to 96 h. From seven BAs only four biogenic amines (tryptamine, putrescine, histamine, and tyramine) were detected while, the cadaverine, spermidine and spermine were absent in all investigated samples. Histamine was the major biogenic amine formed during the storage times and reached the highest concentration of 7.55 ± 0.46 mg/100 g and 17.68 ± 1.30 mg/100 g after 96 h at 4 and 20 °C, respectively. This level of histamine exceeded the maximum tolerance level of 5 mg/100 g. However, the proper icing procedure retarded the histamine effects, resulting only 7.55 mg/100 g after 96 h of ice storage. Spoilage indicator putrescine was only detected after 24-96 h of storage at 4 and 20 °C, respectively. The total volatile base nitrogen and the trimethylamine-nitrogen concentrations were considered to be reliable indicators of freshness index in blue swimmer crab. An aerobic mesophilic plate count of 6.68 and 7.31 log CFU/g were noted for crab stored in ice and ambient temperature after 96 h storage, respectively. It could be concluded that the biogenic amine forming bacteria and other susceptible perishing factors responsible for the biogenic amine formation could be prevented by continuous storage of P. pelagicus at low temperature.

Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties.

The present study reveals purification and characterization of immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus (Pp-Lec). The Pp-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 155 kDa in SDS-PAGE. The surface morphology of purified Pp-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 3.3 min was appeared in high performance liquid chromatography (HPLC) and X-ray diffraction (XRD) analysis expresses a single peak at 31.5° which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Pp-Lec exhibits encapsulation activity against sepharose beads and yeast agglutination activity against Saccharomyces cerevisiae. Moreover, the purified Pp-Lec has the ability to agglutinates with the human erythrocytes among tested and which was observed by light microscopy. In addition, purified Pp-Lec showed the broad spectrum of antibacterial activity against Gram-positive Bacillus pumulis, Bacillus thuringiensis, Enterococcus faecalis and Gram negative Citrobacter amalonaticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter murliniae, Citrobacter freundii, Morganella morganii. Antibiofilm potential of purified Pp-Lec against selective Gram-negative bacteria showed the disruption of biofilm architecture at the concentration of 50 µg ml-1.

Population Structure of the Blue Swimmer Crab Portunus pelagicus in Coastal Areas of Malaysia Inferred from Microsatellites.

Chuan Jian Chai, Yuzine Bin Esa, Muhammad Fadhil Syukri Ismail, and Mohd. Salleh Kamarudin (2017) Portunus pelagicus, distributed throughout the Indo-West Paci c region, is one of the large and edible species of blue swimmer crabs. Increasing demand for the frozen and canned crabmeat industry worldwide has now relied mainly on P. pelagicus which in turn generates splendid income for the fisherman communities. In the present study, the population genetic structure of P. pelagicus was examined using six pairs of microsatellite loci. A total of 87 crab samples were collected from five different coastal areas of Malaysia. Genomic DNA was extracted from each sample for polymerase chain reaction (PCR) amplification and fragment analysis. Four out of six microsatellite primers revealed polymorphic loci in P. pelagicus sampled. The number of alleles per locus in P. pelagicus ranged from 14 to 34. Microsatellites analyses indicated low levels of genetic differentiation among the P. pelagicus populations. The average observed heterozygosity (HO = 0.48) obtained was lower than the standard heterozygosity found in most marine populations (H O = 0.79). The high F IS values (mean F IS = 0.4756) and low F ST values (mean F ST = 0.0413) also suggested the existence of inbreeding among different populations of P. pelagicus. In conclusion, this study was able to shed light on the population structure of P. pelagicus in coastal areas of Malaysia.

Presence of gonadotropin-releasing hormone-like peptide in the central nervous system and reproductive organs of the male blue swimming crab, Portunus pelagicus, and its effect on spermatogenesis.

Our previous studies have demonstrated that lamprey gonadotropin-releasing hormone-III (lGnRH-III)-like peptide occurs in the central nervous system (CNS) of decapod crustaceans (Macrobrachium rosenbergii, Penaeus monodon, Portunus pelagicus), and that lGnRH-III is the most potent in stimulating ovarian maturation compared with other GnRH isoforms. In this study, we examined the localization of lGnRH-III-like peptide in the CNS and male reproductive organs of the blue swimming crab by using anti-lGnRH-III as a probe. In the brain, lGnRH-III immunoreactivity (-ir) was detected in neurons of clusters 6, 10, 11, 14/15, 16, and 17 and in many neuropils. In the subesophageal ganglion, lGnRH-III-ir was present in neurons of the dorso-lateral and ventro-medial clusters. In the thoracic ganglia, lGnRH-III-ir was observed in the large-sized neurons between the thoracic neuropils and in the ventromedial cluster of the abdominal ganglia. In the testis, lGnRH-III-ir was detected in nurse cells, hemocytes, spermatids 2, and the outer and inner zones of the acrosomes of spermatozoa. Bioassay showed that lGnRH-III significantly increased the testis-somatic index, the percentage of late stages of seminiferous tubules (stages VII-IX), the diameter of the seminiferous tubules, and the number of BrdU-labeled early germ cells compared with the control groups. Thus, lGnRH-III-like peptide exists in the male crab and possibly enhances germ cell proliferation and maturation in the testes, leading to increased sperm production.

Induced thermal stress on serotonin levels in the blue swimmer crab, Portunus pelagicus.

The temperature of habitat water has a drastic influence on the behavioral, physiological and biochemical mechanisms of crustaceans. Hyperglycemia is a typical response of many aquatic animals to harmful physical and chemical environmental changes. In crustaceans increased circulating crustacean hyperglycemic hormone (CHH) and hyperglycemia are reported to occur following exposure to several environmental stress. The biogenic amine, serotonin has been found to modulate the CHH levels and oxidation of serotonin into its metabolites is catalysed by monoamine oxidase. The blue swimmer crab, Portunus pelagicus is a dominant intertidal species utilized throughout the indo-pacific region and is a particularly important species of Palk bay. It has high nutritional value and delicious taste and hence their requirements of capture and cultivation of this species are constantly increasing. This species experiences varying and increasing temperature levels as it resides in an higher intertidal zone of Thondi coast. The present study examines the effect of thermal stress on the levels of serotonin and crustacean hyperglycemic hormone in the hemolymph of P. pelagicus and analyzes the effect of the monoamine oxidase inhibitor, pargyline on serotonin and CHH level after thermal stress. The results showed increased levels of glucose, CHH and serotonin on exposure to 26 °C in control animals. Pargyline injected crabs showed highly significant increase in the levels of CHH and serotonin on every 2 °C increase or decrease in temperature. A greater CHH level of 268.86±2.87 fmol/ml and a greater serotonin level of 177.69±10.10 ng/ml was observed at 24 °C. This could be due to the effect of in maintaining the level of serotonin in the hemolymph and preventing its oxidation, which in turn induces hyperglycemia by releasing CHH into hemolymph. Thus, the study demonstrates the effect of thermal stress on the hemolymph metabolites studied and the role of pargyline in elevating the levels of serotonin and CHH on thermal stress in the blue swimmer crab, P. pelagicus.