Impact of anthropogenic global hypoxia on the physiological response of bivalves.

Dissolved oxygen (DO) is an important parameter that affects the biology, physiology, and immunology of aquatic animals. In recent decades, DO levels in the global oceans have sharply decreased, partly due to an increase in atmospheric carbon dioxide, temperature, and anthropogenic nutrient loads. Although there have been many reports on the effects of hypoxia on the survival, growth, behavior, and immunity of bivalves, this information has not been well organized. Therefore, this article provides a comprehensive review of the effects of hypoxia on bivalves. In general, hypoxia negatively impacts the food consumption rate and assimilation efficiency, as well as increasing respiration rates in many bivalves. As a result, it reduces the energy allocation for bivalve growth, shell formation, and reproduction. In severe cases, prolonged exposure to hypoxia can result in mass mortality in bivalves. Moreover, hypoxia also has adverse effects on the immunity and response of bivalves to predators, including decreased burial depths, sensitivity to predators, impairment of byssus production, and negatively impacts on the integrity, strength, and composition of bivalve shells. The tolerance of bivalves to hypoxia largely depends on size and species, with larger bivalves being more susceptible to hypoxia and intertidal species being relatively more tolerant to hypoxia. The information in this article is very useful for elucidating the current research status of hypoxia on bivalves and determining future research directions.

Protein quality of commercially important edible bivalves.

Bivalves are a high-quality source of animal protein for human consumption. In recent years, the demand for bivalve proteins has increased dramatically, leading to a sharp increase in global production of marine bivalves. To date, although the amino acid profiles of many bivalves have been reported, such information has not been well organized. Therefore, there is an urgent need for a comprehensive scientific review of the protein quality of bivalves, especially commercially important edible bivalves. In this context, this study was conducted to evaluate the protein quality of commercially important edible bivalves. In general, most bivalves are rich in protein (> 50% of their dry weight) and amino acids (> 30 g/100g protein). Although most species of bivalves are rich in essential amino acids (EAA) (up to 50 g/100g protein), some species of edible bivalves have very low levels of EAA (< 5 g/100g protein). Based on the AA score, almost all bivalves have at least two limiting AAs. Most bivalve proteins provides delicious flavors with unami, sweetness and a hint of bitterness. The findings of this study not only serve as a a guide for selecting appropriate bivalves based on consumer preferences for specific AAs or AA scores, but also provide information on potential bivalve species for aquaculture to produce higher protein quality to meet the growing demand for high quality animal protein.

Identifying genes involved in the secretory physiological response to feeding in Pacific White Shrimp (Litopenaeus vannamei) using transcriptomics.

The physiological response to feeding is important for production aspects that include feed utilization and growth, and the responses require the action of numerous secretory factors. However, as an important aquaculture animal, the secretory response of Pacific White Shrimp (Litopenaeus vannamei) after feeding has not been comprehensively characterized. In this study, transcriptome analysis showed that 3172 differentially expressed genes were involved in the post-feeding response, including 289 new genes not annotated in the L. vannamei reference genome. Subsequently, 715 differentially expressed secretory reference genes and 18 new differentially expressed secretory genes were obtained through the identification of signal peptides in secreted proteins. Functional classification revealed that differentially expressed secretory genes were enriched in pathways pertaining to lipid metabolism (20 genes), carbohydrate metabolism (21 genes), glycan biosynthesis and metabolism (27 genes), digestive system (40 genes), and transport and metabolism (43 genes). The 14 pathways most enriched by differentially expressed secretory genes involved 83 genes, 71 of which encoded enzymes involved in food digestion and metabolism. Specific enzymes such as lipase 3-like and NPC intracellular cholesterol transporter 1-like in lipid metabolism, alpha-amylase-like and glucosylceramidase-like in carbohydrate metabolism, and cysteine proteinase 4-like and trypsin-1-like in the digestive system were found to be differentially expressed. Furthermore, we discovered a new gene, MSTRG.2504, that participates in the digestive system and carbohydrate metabolism. The study provides valuable insights into the secretory response (especially metabolism-related enzymes) to feeding in L. vannamei, uncovering the significant roles of both known and new genes. Furthermore, this study will improve our understanding of the feeding physiology of L. vannamei and provide a reference basis for further feeding endocrine research in the future.

Identification of key genes and molecular pathways associated with claw regeneration in mud crab (Scylla paramamosain).

The mud crab (Scylla paramamosain) possesses extensive regenerative abilities, enabling it to replace missing body parts, including claws, legs, and even eyes. Studying the genetic and molecular mechanisms underlying regenerative ability in diverse animal phyla has the potential to provide new insights into regenerative medicine in humans. In the present study, we performed mRNA sequencing to reveal the genetic mechanisms underlying the claw regeneration in mud crab. Several differentially expressed genes (DEGs) were expressed in biological pathways associated with cuticle synthase, collagen synthase, tissue regeneration, blastema formation, wound healing, cell cycle, cell division, and cell migration. The top GO enrichment terms were microtubule-based process, collagen trimer, cell cycle process, and extracellular matrix structural constituent. The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. The genes encoding key functional proteins, such as collagen alpha, cuticle protein, early cuticle protein, arthrodial cuticle protein, dentin sialophosphoprotein (DSPP), epidermal growth factor receptor (EGFR), kinesin family member C1 (KIFC1), and DNA replication licensing factor mcm2-like (MCM2) were the most significant and important DEGs suspected to participate in claw regeneration. The findings of this research offer a comprehensive and insightful understanding of the genetic and molecular mechanisms underlying claw regeneration in S. paramamosain. By elucidating the specific genes and molecular pathways implicated in this process, our study contributes significantly to the broader field of regenerative biology and offers potential avenues for further exploration in crustacean limb regeneration.

Identification of key overlapping DEGs and molecular pathways under multiple stressors in the liver of Nile tilapia (Oreochromis niloticus).

The identification of key genes and molecular pathways that are involved in the response to stressors is crucial for controlling stress in fish and sustainable aquaculture. Environmental stressors can induce stress responses in aquatic animals, resulting in compromised immune function, inhibited growth, and increased mortality rates. mRNA-seq analysis provides a powerful tool to identify key genes and pathways associated with stress response. In the present study, mRNA-seq analysis was employed to identify key overlapping differentially expressed genes (DEGs) and molecular pathways under salinity, nitrite, copper, and pH stress in the liver of Nile tilapia (Oreochromis niloticus). The pathways associated with the immune response, oxygen transport, homeostasis, and oxidative stress were enriched across all stressors. The top KEGG pathways were complement and coagulation cascades, PPAR signaling pathway, and cardiac muscle contraction. The top GO enrichment terms were oxidoreductase activity, aerobic respiration, endopeptidase inhibitor activity, endopeptidase regulator activity, heme binding, and iron ion binding. The complement genes (C3, C4, C5, factor B, and factor H), alpha-2-macroglobulin (A2M), hemoglobin subunit epsilon (HBE), hemoglobin subunit alpha (HBA), coagulation factor genes (XI and X) and the cytochrome c oxidase (COX) gene family (cox1, cox2, cox3, cytochrome P450) were identified as key shared genes across multiple stressors. The discovery of these genes and molecular pathways provided a better understanding of the molecular mechanism underlying the stress response in Nile tilapia. The results of the present study can facilitate the development of stress management strategies in Nile tilapia.

Identification of key immune and stress related genes and pathways by comparative analysis of the gene expression profile under multiple environmental stressors in pacific white shrimp (Litopenaeus vannamei).

Water salinity, pH, and nitrite concentration are considered environmental factors affecting the growth rate, survival, health, and physiological conditions of aquatic animals. The identification of key genes that are involved in the response to environmental stressors is essential for controlling stress in aquatic animals and sustainable aquaculture. In this study, RNA sequencing was performed to identify the differentially expressed genes (DEGs) and biological pathways that are involved in the response of the hepatopancreas to environmental stressors, including low salinity stress, nitrite stress, low pH stress, and high pH stress. The DEGs were enriched in biological pathways related to immune response, energy metabolism, oxidative stress response, hemostasis, and enzymatic activity of the hepatopancreas. In addition to the identification of DEGs related to each stressor, some DEGs were found to be expressed among all groups. The most important overlapping DEGs under multiple stressors were juvenile hormone esterase-like protein 2 (JHE-like), myosin light chain, C-type lectin 2, myosin-9-like, anti-lipopolysaccharide factor 1 (ALF-1), peroxisomal acyl-coenzyme An oxidase 1-like (ACX1), hepatic lectin-like, venom phosphodiesterase 2-like, hemolymph clottable protein-like (CP), cathepsin L, and Ras-like protein 2. The results of the present study provide additional information regarding the transcriptional response of the hepatopancreas to low salinity, nitrite, low pH, and high pH stress. Moreover, the discovery of several overlapping DEGs among different stressors provided a better understanding of the molecular function of the hepatopancreas.

Full-Length Transcriptome Reconstruction Reveals the Genetic Mechanisms of Eyestalk Displacement and Its Potential Implications on the Interspecific Hybrid Crab (Scylla serrata ♀ × S. paramamosain ♂).

The lack of high-quality juvenile crabs is the greatest impediment to the growth of the mud crab (Scylla paramamosain) industry. To obtain high-quality hybrid offspring, a novel hybrid mud crab (S. serrata ♀ × S. paramamosain ♂) was successfully produced in our previous study. Meanwhile, an interesting phenomenon was discovered, that some first-generation (F1) hybrid offspring's eyestalks were displaced during the crablet stage I. To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, both single-molecule real-time (SMRT) and Illumina RNA sequencing were implemented. Using a two-step collapsing strategy, three high-quality reconstructed transcriptomes were obtained from purebred mud crabs (S. paramamosain) with normal eyestalks (SPA), hybrid crabs with normal eyestalks (NH), and hybrid crabs with displaced eyestalks (DH). In total, 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in DH. The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant GO terms were related to the cuticle or chitin. Overall, high-quality reconstructed transcriptomes were obtained for the novel interspecific hybrid crab and provided valuable insights into the genetic mechanisms of eyestalk displacement in mud crab (Scylla spp.) crossbreeding.

Evidence from transcriptome analysis unravelled the roles of eyestalk in salinity adaptation in Pacific white shrimp (Litopenaeus vannamei).

Eyestalk is considered the main neuroendocrine organ in crustaceans. Eyestalk regulates reproduction, molting, and energy metabolism by secreting several neurohormones. However, the role of eyestalk in salinity adaptation in crustaceans remains unclear. To reveal the role of eyestalk in salinity adaptation in Litopenaeus vannamei, we performed RNA-seq to compare the transcriptomic response of the eyestalk under low salinity (salinity 3) with that of the control group (salinity 25) for 8 weeks. A total of 479 mRNAs, including 150 upregulated and 329 downregulated mRNAs, were differentially expressed between the two salinity groups. The majority of the differentially expressed genes (DEGs) were enriched in biological pathways related to osmoregulation, metabolism and energy production, and oxidative stress. The most important DEGs associated with osmoregulation were CA4, ATP1A, ATP2B, ABCB1, ABCC4, PhoA, PhoB, NOS1, ACE, ANPEP, and the V-type H+-ATPase E-subunit. The metabolism-related DEGs were divided into three main categories: carbohydrate and energy metabolism (i.e., G6PC, UGT), protein and amino acid metabolism (i.e., SLC15A1, AhcY, GFAT), and lipid and fatty acid metabolism (i.e., GPAT3_4, CYP2J). The key DEGs related to the oxidative stress response were UGT, NDUFB1, QCR7, QCR8, P5CDh, COX6B, and CES1. These results provide evidence for the existence of an eyestalk-salinity adaptation-stress endocrine axis in L. vannamei. These findings provide a better understanding of the molecular mechanism underlying salinity adaptation in L. vannamei.

Whole-transcriptome RNA sequencing revealed the roles of chitin-related genes in the eyestalk abnormality of a novel mud crab hybrid (Scylla serrata ♀ × S. paramamosain ♂).

Chitin is a kind of insoluble structural polysaccharide and plays different roles in different species. In crustaceans, it forms the structural components in the exoskeleton. In our previous studies, novel mud crab hybrids have been produced from the interspecific hybridization of Scylla serrata ♀ × S. paramamosain ♂. Some of the hybrid crabs have been found to be morphologically (eyestalk) abnormal, but the genetic mechanism remains unknown. To address this question, we performed whole-transcriptome RNA sequencing on the control group (normal hybrids), abnormal hybrids, and S. paramamosain to uncover the genetic basis underlying this morphological abnormality. A total of 695 mRNAs, 10 miRNAs, 44 circRNAs, and 1957 lncRNAs were differentially expressed between normal and abnormal hybrids. Several differentially expressed genes (DEGs) associated with chitin and cuticle metabolism were identified, including chitin synthase, chitinase, chitin deacetylase, ß-N-acetylglucosaminidase, ß-1,4-endoglucanase, N-alpha-acetyltransferase, cuticle proprotein, early cuticle protein, and arthrodial cuticle protein. Functional analysis showed that DE miRNAs, DE circRNAs, DE lncRNAs, and lncRNA/circRNA-miRNA-mRNA network were enriched in pathways related to the amino acid, carbohydrate, and glycogen metabolism. Considering the importance of the chitin and cuticle in exoskeleton formation, it can be concluded that the changes in the chitin and cuticle biosynthesis might have caused the eyestalk abnormality in hybrid crabs. These findings can lay the solid foundation for a better understanding of the important roles of chitin and cuticle related genes and the development of hybridization techniques in crustaceans.

The role of the renin-angiotensin system (RAS) in salinity adaptation in Pacific white shrimp (Litopenaeus vannamei).

The renin-angiotensin system (RAS) is a hormonal system that plays an important role in the regulation of blood pressure and cardiovascular homeostasis in mammals. In fishes, the RAS pathway participates in osmoregulation and salinity adaptation. However, the role of the RAS pathway in invertebrates, particularly in crustaceans, remains unknown. In this study, four key genes of the RAS pathway (LV-ACE, LV-APN, LV-AT1R, and LV-RR) were cloned, characterized, and their expression levels were detected in the eyestalk, hepatopancreas, and muscle of Litopenaeus vannamei during long-term and short-term low salinity stress. The results showed that LV-ACE, LV-APN, LV-AT1R, and LV-RR encode 666, 936, 175, and 323 amino acids, respectively. Low salinity stress downregulated the expression levels of LV-ACE, LV-APN, LV-AT1R, and LV-RR in L. vannamei, indicating that the RAS pathway was suppressed under low salinity. Moreover, these genes play important roles in the regulation of drinking rate, controlling urine output, blood glucose, and blood pressure, indicating that their downregulation probably affected the homeostasis of shrimps. These findings provide novel insights into the mechanism of salinity adaptation in L. vannamei.

The significant sex-biased expression pattern of Sp-Wnt4 provides novel insights into the ovarian development of mud crab (Scylla Paramamosain).

The wingless-type MMTV integration site family member-4 (Wnt4), a member of the wingless-related integration site (Wnt) family, is widely accepted as a key regulator of ovarian development in mammals. In this study, a full-length cDNA of Wnt4 (designated as Sp-Wnt4) was cloned, characterized, and functionally studied in mud crab (Scylla paramamosain). The full-length cDNA of Sp-Wnt4 consists of 2659 bp with an open reading frame (ORF) encoding 359 amino acids, a 907 bp 5'-UTR and a 672 bp 3'-UTR. Sp-Wnt4 contains 25 cysteine (Cys) residues and three potential N-glycosylation sites. Sp-Wnt4 protein shared the highest identity (98.9%) to the Wnt4 protein of Portunus trituberculatus. The phylogenetic tree showed that Sp-Wnt4 and Wnt4 protein of Malacostracan crustaceans clustered together, indicating that they had a close genetic distance. Sp-Wnt4 was expressed at a higher level in the ovary compared to other tissues, with the highest expression level at the third stage (O-III) of the ovarian development (P < 0.05). A downward trend was observed in the expression level of Sp-Wnt4 from the embryo stage to crablet stages (P < 0.05). After unilateral eyestalk ablation, the expression level of Sp-Wnt4 significantly increased in testis (14-fold) and downregulated (3.1-fold) in the gill (P < 0.05) of females. In situ hybridization (ISH) assay revealed that Sp-Wnt4 transcripts were mainly localized in the cytoplasm of oocyte cells. These findings showed that Sp-Wnt4 play crucial roles in the ovarian development of S. paramamosain. In conclusion, our study provides novel insights into the evolution and roles of the Wnt4 gene.

Photoperiod affects gamete production, and protein and lipid metabolism in male narrow-clawed Crayfish Pontastacus leptodactylus (Eschscholtz, 1823).

Understanding effects of photoperiod on the reproductive efficiency and physiological responses of Pontastacus leptodactylus is important for successful management of the crayfish hatcheries. Spermatozoal number, gonado-somatic index (GSI), hepatosomatic index (HSI), testicular index (TI), vas deferens index (VDI), and total protein, amino and fatty acid profiles of the hepatopancreas were evaluated in male narrow-clawed crayfish P. leptodactylus. Animals (30-70 g) were maintained utilizing five different photoperiodic regimens (3 L:21D, 6L:18D, 24 L:0D, 0L:24D, and natural photoperiod (9 L:15D)) for 5 weeks. Different photoperiods affected spermatozoal number, GSI, and total protein, amino acid and fatty acid profiles of the hepatopancreas (P < 0.05). The greatest spermatozoal number and GSI were recorded in darkness (P < 0.05). There was the least spermatozoal number (3.5 × 106 ± 0.5) and GSI (2 ± 0.1%) when imposing the 9 L and 24 L photoperiodic regimens, respectively. In addition, maintenance in constant light as compared with other lighting regimens resulted in a greater total protein (43.68 ± 4.83 mg/g), phenylalanine (7.23 ± 0.35%), and total single unsaturated fatty acids (ΣMUFA) (48.07 ± 2.30%), but lesser total polyunsaturated fatty acids (ΣPUFA) (23.36 ± 1.07%), serine (1.53 ± 0.02%), Σn-6 (11.24 ± 1.18%), 20:4 (n-6) (2.7 ± 0.14%), and 20:2 (0.67 ± 0.17%) contents in the hepatopancreas (P < 0.05). The results of the present study indicate total darkness increases the spermatozoal production and imposing longer dark periods improved the reproductive efficiency and physical conditions in male P. leptodactylus.

Optimization of the incubation time and temperature for spermatozoa extraction in freshwater crayfish Pontastacus leptodactylus (Eschscholtz, 1823).

Determination and control of spermatozoa quality in crustacean aquaculture is an important issue for successful and controlled reproduction. Investigation of spermatozoa number in spermatophores is a basic and common parameter for determining the reproductive quality in farmed decapods. In the present study, spermatozoa extraction from spermatophores located in the ductus deferens was conducted in Pontastacus leptodactylus using different incubation times and temperatures. The results indicate that the duration of incubation and temperature affected (P < 0.05) spermatozoa extraction. Greater temperatures (40 and 75 °C) resulted in a reduction (P < 0.05) in number of extracted spermatozoa. In contrast, more spermatozoa were extracted when the 4 and 23 °C temperatures were imposed. After 4 h of incubation, the number of extracted spermatozoa were greatest in number at 23 °C. In conclusion, the greater numbers of crayfish spermatozoa can be obtained when the ductus deferens containing spermatophores is incubated at 23 °C for 4 h as compared with other temperatures and incubation durations. The results of present study are useful for assessing spermatozoa quality in aquaculture as well as the extraction of spermatozoa for research purposes.