Efectos del cobre disuelto sobre la supervivencia, estado antioxidante, expresión de la metalotionina-I mRNA y la respuesta fisiológica de las larvas del cangrejo de Shangai, Eriocheir sinensis (Decapoda: Brachyura)

The lethal concentration of water-borne copper in Chinese mitten crab Eriocheir sinensis larvae was tested by exposing the animals to 0, 0.1, 0.2, 0.3, 0.5 and 0.8 mg Cu L-1 at 20°C for 96 h. The 96-h median lethal concentration (LC50) and its corresponding 95% confident interval was estimated on zoea 1 larvae and megalopa larvae, respectively. Acute dissolved copper toxicity was higher for zoea 1 larvae (0.16 mg L-1) than for megalopa larvae (0.21 mg L-1). The antioxidant status, metallothionein-I mRNA expression and physiological response of the crab to copper toxicity was further investigated by exposing the megalopa larvae to 0, 0.08 and 0.16 mg Cu L-1 for 96 h. The superoxide dismutase activity, catalase activity, glutathione S-transferase (GST) activity and lipid peroxidation content of megalopa larvae increased concomitantly with the exposure time and copper concentration. MT-I mRNA expression levels were positively correlated with both the concentration and duration of copper exposure. The oxygen consumption and respiratory quotient of megalopa larvae in response to 0.16 mg L-1 copper were significantly higher than those in the control group after 96 h of exposure (P < 0.05). The results of this study highlight the potential effects of copper as a common stressor in E. sinensis larvae. MT-I and GST appear to be suitable biomarkers of environmental copper exposure stress in E. sinensis larvaeLa concentración letal de cobre disuelto se determina mediante la exposición de larvas del cangrejo de Shangai (Eriocheir sinensis) a dosis de 0, 0.1, 0.2, 0.3, 0.5 y 0.8 mg Cu L–1, a 20°C durante 96 h. La concentración letal media (LC50) y su correspondiente intervalo de confianza del 95% fueron estimados en los estadios larvales zoea I y megalopa. La toxicidad aguda del cobre disuelto fue mayor sobre larvas de zoea I (0.16 mg L–1) que sobre las de megalopa (0.21 mg L–1). El estado antioxidante, la expresión del mRNA de la metalotionina–I y la respuesta fisiológica del cangrejo a la toxicidad del cobre fueron posteriormente investigados exponiendo las larvas de megalopa a dosis de 0, 0.08 y 0.16 mg Cu L–1 durante 96 h. La actividad de la superóxido dismutasa (SOD), de la catalasa (CAT), de la glutatión-S-transferasa (GST) y la peroxidación lipídica de las larvas de megalopa se incrementó en relación con el tiempo de exposición y la concentración de cobre. Los niveles de expresión de MT-1 mRNA se correlacionaron positivamente con la concentración de cobre y el tiempo de exposición. El consumo de oxígeno y la tasa respiratoria de las larvas de megalopa fueron significativamente más elevadas que las del grupo control (P < 0.05) en respuesta a dosis de 0.16 mg Cu L–1, durante 96 h de exposición. Los resultados presentados en este estudio ponen de manifiesto los efectos potenciales del cobre como factor de estrés sobre las larvas de E. sinensis. MT-I y GST parecen ser adecuados biomarcadores de estrés a la exposición de Cu ambiental en larvas de E. sinensis

Accurate RSS-Based Localization Using an Opposition-Based Learning Simulated Annealing Algorithm

Wireless sensor networks require accurate target localization, often achieved through received signal strength (RSS) localization estimation based on maximum likelihood (ML). However, ML-based algorithms can suffer from issues such as low diversity, slow convergence, and local optima, which can significantly affect localization performance. In this paper, we propose a novel localization algorithm that combines opposition-based learning (OBL) and simulated annealing algorithm (SAA) to address these challenges. The algorithm begins by generating an initial solution randomly, which serves as the starting point for the SAA. Subsequently, OBL is employed to generate an opposing initial solution, effectively providing an alternative initial solution. The SAA is then executed independently on both the original and opposing initial solutions, optimizing each towards a potential optimal solution. The final solution is selected as the more effective of the two outcomes from the SAA, thereby reducing the likelihood of the algorithm becoming trapped in local optima. Simulation results indicate that the proposed algorithm consistently outperforms existing algorithms in terms of localization accuracy, demonstrating the effectiveness of our approach

Molecular Cloning, Characterization, and mRNA Expression of Hemocyanin Subunit in Oriental River Prawn Macrobrachium nipponense

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Hemocyanin is a copper-containing protein with immune function against disease. In this study, a hemocyanin subunit named MnHc-1 was cloned from Macrobrachium nipponense. The full-length cDNA of MnHc-1 was 2,163 bp with a 2,028-bp open reading frame (ORF) encoding a polypeptide of 675 amino acids. The MnHc-1 mRNA was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary, and stomach, with the highest level in the hepatopancreas. In the infection trial, the MnHc-1 mRNA transcripts in the hemocytes were significantly downregulated at 3 h after injection of Aeromonas hydrophila and then upregulated at 6 h and 12 h, followed by a gradual recovery from 24 to 48 h. The MnHc-1 transcriptional expression in the hepatopancreas was measured after M. nipponense were fed seven diets with 2.8, 12.2, 20.9, 29.8, 43.1, 78.9, and 157.1 mg Cu kg−1 for 8 weeks, respectively. The level of MnHc-1 mRNA was significantly higher in the prawns fed 43.1–157.1 mg Cu kg−1 diet than in that fed 2.8–29.8 mg Cu kg−1 diet. This study indicated that the MnHc-1 expression can be affected by dietary copper and the hemocyanin may potentially participate in the antibacterial defense of M. nipponense

Identification of Candidate Genes for the Plateau Adaptation of a Tibetan Amphipod, Gammarus lacustris, Through Integration of Genome and Transcriptome Sequencing

The amphipod Gammarus lacustris has been distributing in the Tibetan region with well-known uplifts of the Tibetan plateau. It is hence considered as a good model for investigating stress adaptations of the plateau. Here, we sequenced the whole-genome and full-length transcriptome of G. lacustris, and compared the transcriptome results with its counterpart Gammarus pisinnus from a nearby plain. Our main goal was to provide a genomic resource for investigation of genetic mechanisms, by which G. lacustris adapted to living on the plateau. The final draft genome assembly of G. lacustris was 5.07 gigabases (Gb), and it contained 443,304 scaffolds (&gt;2 kb) with an N50 of 2,578 bp. A total of 8,858 unigenes were predicted in the full-length transcriptome of G. lacustris, with an average gene length of 1,811 bp. Compared with the G. pisinnus transcriptome, 2,672 differentially expressed genes (DEGs) were up-regulated and 2,881 DEGs were down-regulated in the G. lacustris transcriptome. Along with these critical DEGs, several enriched metabolic pathways, such as oxidative phosphorylation, ribosome, cell energy homeostasis, glycolysis and gluconeogenesis, were predicted to play essential roles in the plateau adaptation. In summary, the present study provides a genomic basis for understanding the plateau adaption of G. lacustris, which lays a fundamental basis for further biological and ecological studies on other resident aquatic species in the Tibetan plateau

Molecular Cloning and Expression Analysis of Lactate Dehydrogenase from the Oriental River Prawn Macrobrachium nipponense in Response to Hypoxia

Metabolic adaption to hypoxic stress in crustaceans implies a shift from aerobic to anaerobic metabolism. Lactate dehydrogenase (LDH) is a key enzyme in glycolysis in prawns. However, very little is known about the role of LDH in hypoxia inducible factor (HIF) pathways of prawns. In this study, full-length cDNA of LDH (MnLDH) was obtained from the oriental river prawn Macrobrachium nipponense, and was characterized. The full-length cDNA is 2267-bp with an open reading frame of 999 bp coding for a protein of 333 amino acids with conserved domains important for function and regulation. Phylogenetic analysis showed that MnLDH is close to LDHs from other invertebrates. Quantitative real-time PCR revealed that MnLDH is expressed in various tissues with the highest expression level in muscle. MnLDH mRNA transcript and protein abundance in muscle, but not in hepatopancreas, were induced by hypoxia. Silencing of hypoxia-inducible factor 1 (HIF-1) &alpha; or HIF-1&beta; subunits blocked the hypoxia-dependent increase of LDH expression and enzyme activity in muscle. A series of MnLDH promoter sequences, especially the full-length promoter, generated an increase in luciferase expression relative to promoterless vector; furthermore, the expression of luciferase was induced by hypoxia. These results demonstrate that MnLDH is probably involved a HIF-1-dependent pathway during hypoxia in the highly active metabolism of muscle

Molecular Cloning and Functional Characterization of a Hexokinase from the Oriental River Prawn Macrobrachium nipponense in Response to Hypoxia

Metabolic adjustment to hypoxia in Macrobrachium nipponense (oriental river prawn) implies a shift to anaerobic metabolism. Hexokinase (HK) is a key glycolytic enzyme in prawns. The involvement of HK in the hypoxia inducible factors (HIFs) pathway is unclear in prawns. In this study, the full-length cDNA for HK (MnHK) was obtained from M. nipponense, and its properties were characterized. The full-length cDNA (2385 bp) with an open reading frame of 1350 bp, encoded a 450-amino acid protein. MnHK contained highly conserved amino acids in the glucose, glucose-6-phosphate, ATP, and Mg+2 binding sites. Quantitative real-time reverse transcription PCR assays revealed the tissue-specific expression pattern of MnHK, with abundant expression in the muscle, and gills. Kinetic studies validated the hexokinase activity of recombinant HK. Silencing of HIF-1α or HIF-1β subunit genes blocked the induction of HK and its enzyme activities during hypoxia in muscles. The results suggested that MnHK is a key factor that increases the anaerobic rate, and is probably involved in the HIF-1 pathway related to highly active metabolism during hypoxia

Hypoxia Induces Changes in AMP-Activated Protein Kinase Activity and Energy Metabolism in Muscle Tissue of the Oriental River Prawn Macrobrachium nipponense

Hypoxia has important effects on biological activity in crustaceans, and modulation of energy metabolism is a crucial aspect of crustaceans’ ability to respond to hypoxia. The adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) enzyme is very important in cellular energy homeostasis; however, little information is known about the role of AMPK in the response of prawns to acute hypoxia. In the present study, three subunits of AMPK were cloned from the oriental river prawn, Macrobrachium nipponense. The full-length cDNAs of the α, β, and γ AMPK subunits were 1,837, 3,174, and 3,773 bp long, with open reading frames of 529, 289, and 961 amino acids, respectively. Primary amino acid sequence alignment of these three subunits revealed conserved similarity between the functional domains of the M. nipponense AMPK protein with AMPK proteins of other animals. The expression of the three AMPK subunits was higher in muscle tissue than in other tissues. Furthermore, the mRNA expression of AMPKα, AMPKβ, and AMPKγ were significantly up-regulated in M. nipponense muscle tissue after acute hypoxia. Probing with a phospho-AMPKα antibody revealed that AMPK is phosphorylated following hypoxia; this phosphorylation event was found to be essential for AMPK activation. Levels of glucose and lactic acid in hemolymph and muscle tissue were significantly changed over the course of hypoxia and recovery, indicating dynamic changes in energy metabolism in response to hypoxic stress. The activation of AMPK by hypoxic stress in M. nipponense was compared to levels of muscular AMP, ADP, and ATP, as determined by HPLC; it was found that activation of AMPK may not completely correlate with AMP:ATP ratios in prawns under hypoxic conditions. These findings confirm that the α, β, and γ subunits of the prawn AMPK protein are regulated at the transcriptional and protein levels during hypoxic stress to facilitate maintenance of energy homeostasis

Based on the Metabolomic Approach the Energy Metabolism Responses of Oriental River Prawn Macrobrachium nipponense Hepatopancreas to Acute Hypoxia and Reoxygenation

Hypoxia represents a major physiological challenge for prawns and is a problem in aquaculture. Therefore, an understanding of the metabolic response mechanism of economically important prawn species to hypoxia and re-oxygenation is essential. However, little is known about the intrinsic mechanisms by which the oriental river prawn Macrobrachium nipponense copes with hypoxia at the metabolic level. In this study, we conducted gas chromatography-mass spectrometry-based metabolomics studies and assays of energy metabolism-related parameters to investigate the metabolic mechanisms in the hepatopancreas of M. nipponense in response to 2.0 O2/L hypoxia for 6 and 24 h, and reoxygenation for 6 h following hypoxia for 24 h. Prawns under hypoxic stress displayed higher glycolysis-related enzyme activities and lower mRNA expression levels of aerobic respiratory enzymes than those in the normoxic control group, and those parameters returned to control levels in the reoxygenated group. Our results showed that hypoxia induced significant metabolomic alterations in the prawn hepatopancreas within 24 h. The main metabolic alterations were depletion of amino acids and 2-hydroxybutanoic acid and accumulation of lactate. Further, the findings indicated that hypoxia disturbed energy metabolism and induced antioxidant defense regulation in prawns. Surprisingly, recovery from hypoxia (i.e., reoxygenation) significantly affected 25 metabolites. Some amino acids (valine, leucine, isoleucine, lysine, glutamate, and methionine) were markedly decreased compared to the control group, suggesting that increased degradation of amino acids occurred to provide energy in prawns at reoxygenation conditions. This study describes the acute metabolomic alterations that occur in prawns in response to hypoxia and demonstrates the potential of the altered metabolites as biomarkers of hypoxia