The effects of ammonia stress exposure on protein degradation, immune response, degradation of nitrogen-containing compounds and energy metabolism of Chinese mitten crab.

BACKGROUND: The Chinese mitten crab is one of the most economically important crabs that are widely farmed in China. Ammonia, which is a main physiological challenge for crab culture, grows rapidly in the intensive culture system over time, but little information is available with Chinese mitten crab on the molecular mechanisms. METHODS AND RESULTS: Therefore, to understand the mechanism of response to ammonia stress in Eriocheir japonica sinensis, comparative transcriptome analysis was used to identify the key genes and pathways in hepatopancreas challenged with ammonia stress (325.07 mg/L NH4Cl). By sequencing the transcriptome hepatopancreas of E. j. sinensis treated with ammonia, 366,472 unigenes were obtained and annotated into several public libraries for later analyses. Subsequently, 1775 differentially expressed genes (DEGs) were identified according to comparative transcriptome analysis, of which 307 were up-regulated and 1468 were down-regulated. According to the DEGs of GO and KEGG enrichment analyses, we focused on four aspects of significant enrichment in this study: protein degradation, immune response, degradation of nitrogen-containing compounds and energy metabolism. The genes involved in protein degradation and energy metabolism process showed a significant decrease which was consisting of overall biological activity of E. j. sinensis decreased. In addition, five genes involved in high concentration of ammonia were discovered and validated by qRT-PCR. CONCLUSIONS: This study will help us understand the molecular mechanisms of E. j. sinensis under high ammonia exposure and provide valuable information to the future research of other crabs with ammonia exposure.

Comparative transcriptome analysis of the gills of Procambarus clarkii provide novel insights into the response mechanism of ammonia stress tolerance.

Procambarus clarkii is an important model crustacean organism in many researches. Ammonia nitrogen is one of common contaminants in aquatic environment, influencing the health of aquatic organisms. The primary objective of this study was to investigate molecular mechanisms on ammonia stress in gills of P. clarkii to provide new insights into the strategies of aquatic animals in responding to high concentration of ammonia in the environment. Procambarus clarkii were randomly assigned into two groups (ammonia stress group, AG; control group, CG), and gill samples were dependently excised from AG and CG. Then response mechanisms on ammonia stress were investigated based on transcriptome data of P. clarkii. 9237 differentially expressed genes were identified in ammonia stress group. The genes of ion transport enzymes (NKA and SLC6A5S) were significantly up-regulated. Whereas the immune-related genes (e.g. MAP3K7, HSP70, HSP90A, CTSF, CTSL1, CHI and CTL4) and pathways were significantly up-regulated, which played an important role in reacting to ammonia stress. Procambarus clarkii may enhance immune defense to counteract ammonia toxicity by the up-regulation of immune-related genes and signaling pathways. The activities of ion transport enzymes are changed to mobilise signal transduction and ion channel regulation for adapting to ammonia environment. These previous key genes play an important role in resistance to ammonia stress to better prepare for survival in high concentration of ammonia.

Toxic effects of metal copper stress on immunity, metabolism and pathologic changes in Chinese mitten crab (Eriocheir japonica sinensis).

Copper (Cu2+), which represents a major physiological challenge for crab culture, is ubiquitous in the aquatic culture environment, and gills are the first organs that come into direct contact with the environment. However, the molecular basis of the response of crabs to Cu2+ stress remains unclear. Here, we conducted a transcriptome and differential expression analysis on the gills from Chinese mitten crab unexposed and exposed to Cu2+ for 24 h. The comparative transcriptome analysis identified 2486 differentially expressed genes (DEGs). GO functional analysis and KEGG pathway analysis revealed some DEGs, which were mostly related to immunity, metabolism, osmotic regulation, Cu2+ homeostasis regulation, antioxidant activity, and detoxification process. Some pathways related to humoral and cellular immunity, such as phagosome, peroxisome, lysosome, mTOR signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, and T cell receptor signaling pathway were enhanced under Cu2+ stress. In addition, Cu2+ stress altered the expression patterns of key phagocytosis and apoptosis genes (lectin, cathepsin L, Rab7, and HSP70), confirming that Cu2+ can induce oxidative stress and eventually even apoptosis. Histological analysis revealed that the copper can induce damage at the cellular level. This comparative transcriptome analysis provides valuable molecular information to aid future study of the immune mechanism of Chinese mitten crab in response to Cu2+ stress and provides a foundation for further understanding of the effects of metal toxicity.

Comparative transcriptome analysis of the gills of Cardisoma armatum provides novel insights into the terrestrial adaptive related mechanism of air exposure stress.

Cardisoma armatum is a typical member of the Gecarcinidae which show significant behavioral, morphological, physiological, and/or biochemical adaptations permitting extended activities on the land. The special gills (branchiostegal lung) of C. armatum play an important role in maintaining osmotic pressure balance and obtaining oxygen to adapt to the terrestrial environment. However, adaptive molecular mechanisms responding to air exposure in C. armatum are still poorly understood. In this study, transcriptomic analysis and histological analysis were conducted on the gills to test adaptive capabilities over 8 h between the aerial exposure (AE) and the water immersion (WI) group. Differentially expressed genes (DEGs) related to terrestrial adaptation were categorized into four broad categories: ion transport, acid-base balance, energy metabolism and immune response. This is the first research to reveal the molecular mechanism of terrestrial adaptation in C. armatum, and will provide new insight into the molecular genetic basis of terrestrial adaptation in crabs.

Surface Properties and Denitrification Performance of Impurity-Removed Rare Earth Concentrate.

Acid leaching and alkali roasting were used to remove impurities such as Ca and Si in Baiyun Obo rare earth concentrate. The effects of acid-base treatment on the physical and chemical properties of the samples were analyzed by scanning electron microscopy, X-ray diffraction, Brunauer-Emmett-Teller characterization, X-ray photoelectron spectroscopy, H2-temperature-programmed reduction, NH3-temperature-programmed desorption (TPD), and NO-TPD. Results showed that the content of Ce7O12 in the rare earth concentrates increased and the dispersion was uniform. The grains became smaller, the specific surface area of rare earth concentrates increased, and the active sites were more exposed. Ce coexisted in the form of Ce3+ and Ce4+, whereas Fe coexisted in the form of Fe3+ and Fe2+. The content of Fe3+ was increased. The acid-base-treated rare earth concentrates had a denitration efficiency of 87.4% at a reaction temperature of 400 °C.

Characterization of the complete mitochondrial genome of Uca lacteus and comparison with other Brachyuran crabs.

Brachyuran crabs comprise the most species-rich clade among the crustacean order Decapoda and are divided into several major superfamilies. However, the monophyly of the superfamilies Ocypodoidea and Grapsoidea in their current compositions within the Brachyura remains inconclusive. In this study, the complete mitochondrial genome (mitogenome) of Uca lacteus (Ocypodoidea, Ocypodidae) was sequenced, annotated, and compared with those of other Brachyuran crabs. The circular mitogenome of U. lacteus is 15,661 base pairs long and contains the entire set of 37 genes and an A + T-rich region typically observed in decapod mitogenomes. Secondary structures of several tRNAs are partly missing (trnS1), and the number of bases is significantly decreased (trnD and trnF), as discovered in many other metazoans. We compared the gene order of U. lacteus with other species of Ocypodidae and found that they are consistent. The gene rearrangement of Ocypodidae is also identical to that of the ancestor of Brachyura. However, the order of the trnH gene varies from the rearrangement of ancestral Decapoda. Accordingly, we hypothesized that this rearrangement of trnH underwent a translocation during the evolution from Decapoda to Brachyura. The phylogenetic relationship of the 81 Brachyura species and one outgroup was recovered based on 13 protein-coding genes. This analysis confirmed that U. lacteus belongs to the family Ocypodidae and established a paraphyletic relationship between Ocypodoidea and Grapsoidea.

Comparative transcriptome analysis of the gills of Procambarus clarkii provides novel insights into the immune-related mechanism of copper stress tolerance.

The red-swamp crayfish (Procambarus clarkii) is the most important economic shrimp species in China, and is an important model crustacean organism in many fields of research. In crustaceans, gills interface directly with the ambient environment and thus play a vital role in the toxicology. In the context of increasing environmental heavy metal pollution, the relationship between copper (Cu2+) stress and the immune response of P. clarkii has recently received considerable attention. However, impact of Cu2+ on the crayfish immune system is still not fully understood. In this study, we used Illumina sequencing technology to perform a transcriptome analysis of the gills of P. clarkii after 24 h of Cu2+ treatment. A total of 37,226,812 unigenes were assembled, and 1943 unigenes were significantly differentially expressed between the control and Cu2+ treatment groups. Functional categorization of differentially expressed genes (DEGs) revealed that genes related to antioxidant activity, detoxication, metabolic processes, biosynthetic processes, and immune system processes were differentially regulated during Cu2+ stress. In addition, DEGs in the immune system were classified as being related to the MAPK signaling pathway, purine metabolism, Toll and Imd signaling pathway, PI3K-Akt signaling pathway and Hippo signaling pathway. Five genes (CuZnSOD, CAT, IDH1, PHYH and DECR2) were significantly up-regulated in the peroxisome pathway, which plays an important role in reacting to oxidative stress. Importantly, qRT-PCR validation of the results for seven genes chosen at random (NDK, ATP6L, ATP5C1, RPS14, RPL22e, CTSF and HSP90A) confirmed the Illumina sequencing results. This study provides a valuable starting point for further studies to elucidate the molecular basis of the immune system's response to Cu2+ stress in crayfish.

The complete mitochondrial genome of Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) and comparison with other Brachyuran crabs.

Mitochondrial DNA (mtDNA) is an extrachromosomal genome which can provide important information for evolution and phylogenetic analysis. In this study, we assembled a complete mitogenome of a crab Parasesarma pictum (Brachyura: Grapsoidea: Sesarmidae) from next generation sequencing reads at the first time. P. pictum is a mudflat crab, belonging to the Sesarmidae family (subfamily Sesarminae), which is perched on East Asia. The 15,716 bp mitogenome covers 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and one control region (CR). The control region spanns 420 bp. The genome composition was highly A+T biased 75.60% and showed negative AT-skew (-0.03) and negative GC-skew (-0.47). Compared with the ancestor of Brachyura, the gene order of Sesarmidae has several differences and the gene order of P. pictum is typical for mitogenomes of Sesarmidae. Phylogenetic tree based on nucleotide sequences of mitochondrial 13 PCGs using BI and ML determined that P. pictum has a sister group relationship with Parasesarma tripectinis and belongs to Sesarmidae.

The complete mitochondrial genomes of Tarsiger cyanurus and Phoenicurus auroreus: a phylogenetic analysis of Passeriformes.

Passeriformes is the largest group within aves and the phylogenetic relationships between Passeriformes have caused major disagreement in ornithology. Particularly, the phylogenetic relationships between muscicapoidea and sylvioidea are complex, and their taxonomic boundaries have not been clearly defined. Our aim was to study the status of two bird species: Tarsiger cyanurus and Phoenicurus auroreus. Furthermore, we analyzed the phylogenetic relationships of Passeriformes. Complete mitochondrial DNA (mtDNA) sequences of both species were determined and the lengths were 16,803 (T. cyanurus) and 16,772 bp (P. auroreus), respectively. Thirteen protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region were identified in these mtDNAs. The contents of A and T at the base compositions was significantly higher than the content of G and C, and this AT skew was positive, while the GC skew was negative. The monophyly of Passeriformes is divided into four major clades: Corvoidea, Sylvioidea, Passeroidea, and Musicicapoidea. Paridae should be separated from the superfamily Sylvioidea and placed within the superfamily Muscicapoidea. The family Muscicapidae and Corvida were paraphyly, while Carduelis and Emberiza were grouped as a sister taxon. The relationships between some species of the order passeriformes may remain difficult to resolve despite an effort to collect additional characters for phylogenetic analysis. Current research of avian phylogeny should focus on adding characters and taxa and use both effectively to obtain a better resolution for deeper and shallow nodes.

Complete mitochondrial genome of Parasesarma affine (Brachyura: Sesarmidae): Gene rearrangements in Sesarmidae and phylogenetic analysis of the Brachyura.

In this study, the complete mitochondrial DNA (mtDNA) sequence of the crab Parasesarma affine is determined, characterized and compared with other decapod crustaceans. The P. affine mitochondrial genome (mitogenome) is 15,638 bp in size, and contains 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes and a control region (CR). Then, 23 of the 37 genes are encoded by the heavy (+) strand while 14 are encoded by the light (-) strand. All PCGs are initiated by ATN codons and 4 of the 13 PCGs harbored the incomplete termination codon by T or TA. The CR with a high A + T% (82.33%) spans 678 bp. The nucleotide composition of the P. affine mitogenome is also biased toward A + T nucleotides (74.83%). The gene order of P. affine has a difference that trnI-trnQ turns into trnQ-trnI when compared with ancestor of Brachyura, which can also been seen in other Sesarmidae species. Phylogenetic tree based on nucleotide sequences of mitochondrial 13 PCGs from 49 decapod crustaceans and one outgroup using Bayesian inference (BI) and Maximum Likelihood (ML), which determined that P. affine belongs to Sesarmidae and Parasesarma is monophyletic.

Adaptive evolution of osmoregulatory-related genes provides insight into salinity adaptation in Chinese mitten crab, Eriocheir sinensis.

Osmoregulation is an important mechanism by which euryhaline crustaceans regulate osmotic and ionic concentrations. The Chinese mitten crab (Eriocheir sinensis) is a strong osmoregulating animal model among crustacean species, as it can maintain its hemolymph composition and survives well in either seawater or freshwater. Osmoregulation by E. sinensis during physiological adaptation has been studied extensively. However, the genetic basis of osmoregulation in E. sinensis for acclimating to changing salinities remains unclear. The current study investigated five genes involved in E. sinensis osmoregulation and compared them with a representative marine crab Portunus trituberculatus to test whether adaptive evolution has occurred changing salinity conditions. The results showed that carbonic anhydrase (CA), cytochrome P450 4C (CYP4C), glutamate dehydrogenase (GDH), and the Na+/H+ exchanger (NHE) have undergone positive selection (i.e., directional selection) in E. sinensis. Thus, the positive selection in CA and NHE suggests that E. sinensis has enhanced capacity for maintaining systemic acid-base balance and ion regulation. GDH and CYP4C also demonstrated positive selection in E. sinensis, suggesting that E. sinensis might have acquired an enhanced capacity to metabolize glutamate and synthesize ecdysteroids in response to a change in osmotic concentration. The present study provides new insight into the molecular genetic basis of salinity adaption in E. sinensis.

Next-generation sequencing yields the complete mitogenome of Caridina multidentata and phylogenetic analysis.

In this study, the complete mitochondrial genome (mitogenome) of Caridina multidentata is reported for the first time. These data demonstrate that the C. multidentata mitochondrial genome is a 15,825 bp circular molecule and encodes the typical 37 metazoan mitochondrial genes [13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs)] and an A + T-rich control region. The genome composition was highly A + T biased 63.45% and showed negative AT-skew (-0.131) and slightly possitive GC-skew (0.018). The complete mitogenome provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis for shrimps.