Chromosome-level genome assembly of Paralithodes platypus provides insights into evolution and adaptation of king crabs.

The blue king crab, Paralithodes platypus, which belongs to the family Lithodidae, is a commercially and ecologically important species. However, a high-quality reference genome for the king crab has not yet been reported. Here, we assembled the first chromosome-level blue king crab genome, which contains 104 chromosomes and an N50 length of 51.15 Mb. Furthermore, we determined that the large genome size can be attributed to the insertion of long interspersed nuclear elements and long tandem repeats. Genome assembly assessment showed that 96.54% of the assembled transcripts could be aligned to the assembled genome. Phylogenetic analysis showed the blue king crab to have a close relationship with the Eubrachyura crabs, from which it diverged 272.5 million years ago. Population history analyses indicated that the effective population of the blue king crab declined sharply and then gradually increased from the Cretaceous and Neogene periods, respectively. Furthermore, gene families related to developmental pathways, steroid and thyroid hormone synthesis, and inflammatory regulation were expanded in the genome, suggesting that these genes contributed substantially to the environmental adaptation and unique body plan evolution of the blue king crab. The high-quality reference genome reported here provides a solid molecular basis for further study of the blue king crab's development and environmental adaptation.

Evolution of digestive enzyme genes associated with dietary diversity of crabs.

Crabs feed on a wide range of items and display diverse feeding strategies. The primary objective of this study was to investigate 10 digestive enzyme genes in representative crabs to provide insights into the genetic basis of feeding habits among crab functional groups. Crabs were classified into three groups based on their feeding habits: herbivores (HV), omnivores (OV), and carnivores (CV). To test whether crabs' feeding adaptations matched adaptive evolution of digestive enzyme genes, we examined the 10 digestive enzyme genes of 12 crab species based on hepatopancreas transcriptome data. Each of the digestive enzyme genes was compared to orthologous sequences using both nucleotide- (i.e., PAML and Datamonkey) and protein-level (i.e., TreeSAAP) approaches. Positive selection genes were detected in HV crabs (AMYA, APN, and MGAM) and CV crabs (APN, CPB, PNLIP, RISC, TRY, and XPD). Additionally, a series of positive selection sites were localized in important functional regions of these digestive enzyme genes. This is the first study to characterize the molecular basis of crabs' digestive enzyme genes based on functional feeding group. Our data suggest that HV crabs have evolved an enhanced digestion capacity for carbohydrates, and CV crabs have acquired digestion capacity for proteins and lipids.

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.

Comparative mitochondrial genomic analysis of Macrophthalmus pacificus and insights into the phylogeny of the Ocypodoidea & Grapsoidea.

Grapsoidea and Ocypodoidea, two of the most abundant and economically important groups in Brachyura, are of great commercial value to fisheries and aquaculture. However, the taxonomy of Ocypodoidea and Grapsoidea has long been highly disputed. Previous studies have investigated this problem through phylogenetic analysis based on limited taxonomic sampling, with different reports proposing either monophyly or paraphyly, but no definitive conclusion has been reached. In this study, the complete mitogenome of Macrophthalmus pacificus (Ocypodoidea, Macrophthalmidae) is reported on and the relationship between Ocypodoidea and Grapsoidea is further investigated. Sequencing the M. pacificus mitogenome, which is a closed circular molecule containing a typical 37 genes, preliminarily determined the ancestral gene order of Macrophthalmidae, which is consistent with previous studies. Comparative analyses of gene order among Ocypodoidea and Grapsoidea revealed that Varunidae (Grapsoidea) and Macrophthalmidae (Ocypodoidea) have the same rearrangement, which confirms previous research. Larger data analysis revealed that these two families (Varunidae and Macrophthalmidae) cluster into a monophyletic clade as sister groups. Rearrangement and phylogeny lines of evidence is concluded that Varunidae and Macrophthalmidae may be of common origin. Furthermore, the remaining Ocypodoidea and Grapsoidea families mix paraphyletically in the phylogenetic tree. Therefore, both gene rearrangement and phylogenetic analysis support the paraphyly of Ocypodoidea and Grapsoidea, which reinforces this view. These findings provide important information regarding Brachyura's phylogenetic relationships, which demonstrates the advantage of mitogenome sequence data in phylogenetic studies.

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 in the hepatopancreas of Helice tientsinensis exposed to the toxic metal cadmium.

BACKGROUND: The mudflat crab Helice tientsinensis is one of the most commercially valuable species for crabmeat production due to its delicious taste. These crabs are mainly found in coastal wetland where they are seriously threatened by toxic heavy metal pollution. In crustaceans, the hepatopancreas is an important organ for detoxification, and metal toxic substances can be converted to non-toxic or less toxic compounds in this organ. OBJECTIVE: To develop a better understanding of the molecular response of H. tientsinensis to the toxic metal cadmium (Cd) and provide a molecular basis for the toxic metal tolerance of H. tientsinensis. METHODS: In this study, we performed comparative hepatopancreas transcriptome analysis between H. tientsinensis unexposed (as control) and exposed to the toxic metal Cd for 48 h. RESULTS: We identified 1089 Cd stress significantly-upregulated and 1560 Cd stress significantly-downregulated unigenes. Functional categorization and annotation of these differentially-expressed genes (DEGs) demonstrated that the response to Cd stress in the hepatopancreas of H. tientsinensis mainly involves "antioxidant activity", "detoxification", "toxin degradation activity" and "immune system process". In addition, five genes (ABCC1, NDUFAF5, ASTL, DES1, CYP27A) were identified as possible major targets for toxic metal tolerance. CONCLUSION: This is the first time reporting that the response of H. tientsinensis to Cd exposure at the transcriptome level, and it lays the foundation for understanding the molecular mechanisms of the response of H. tientsinensis to environmental toxic metal stress.

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.

Mitochondrial OXPHOS genes provides insights into genetics basis of hypoxia adaptation in anchialine cave shrimps.

Cave shrimps from the genera Typhlatya, Stygiocaris and Typhlopatsa (TST complex) comprises twenty cave-adapted taxa, which mainly occur in the anchialine environment. Anchialine habitats may undergo drastic environmental fluctuations, including spatial and temporal changes in salinity, temperature, and dissolved oxygen content. Previous studies of crustaceans from anchialine caves suggest that they have possessed morphological, behavioral, and physiological adaptations to cope with the extreme conditions, similar to other cave-dwelling crustaceans. However, the genetic basis has not been thoroughly explored in crustaceans from anchialine habitats, which can experience hypoxic regimes. To test whether the TST shrimp-complex hypoxia adaptations matched adaptive evolution of mitochondrial OXPHOS genes. The 13 OXPHOS genes from mitochondrial genomes of 98 shrimps and 1 outgroup were examined. For each of these genes was investigated and compared to orthologous sequences using both gene (i.e. branch-site and Datamonkey) and protein (i.e. TreeSAAP) level approaches. Positive selection was detected in 11 of the 13 candidate genes, and the radical amino acid changes sites scattered throughout the entire TST complex phylogeny. Additionally, a series of parallel/convergent amino acid substitutions were identified in mitochondrial OXPHOS genes of TST complex shrimps, which reflect functional convergence or similar genetic mechanisms of cave adaptation. The extensive occurrence of positive selection is suggestive of their essential role in adaptation to hypoxic anchialine environment, and further implying that TST complex shrimps might have acquired a finely capacity for energy metabolism. These results provided some new insights into the genetic basis of anchialine hypoxia adaptation.

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.

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.

The complete mitogenome of Metopograpsus quadridentatus and phylogenetic analysis.

The complete mitochondrial genome of Metopograpsus quadridentatus was determined to be 15,520 bp in length. It consists of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs and a control region. There are 13 overlapping regions in the genome with 1 to 25 bp length. The largest overlapping region is located between nad1 and trnL1. The AT-skew and GC-skew for the whole mitogenome are both negative, indicating a higher occurrence of Ts than As and Cs than Gs. The molecular data here we presented could play a useful role to study the evolutionary relationships and population genetics of Grapsidae crabs.

Inhibition effects of benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol on the activity of mushroom tyrosinase.

Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin synthesis and fruit-vegetable browning. The inhibition of benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol on mushroom tyrosinase was first investigated. The results shown that these three compounds could effectively inhibit the enzyme activity sharply and the inhibitory effects were determined to be reversible. Their inhibitor concentrations leading to 50% activity lost values were determined to be 1.5, 2.8, and 1.1 mM for monophenolase and 2.0, 0.6, and 0.8 mM for diphenolase, respectively. For the monophenolase activity, all of these three compounds were mixed-type inhibitors, however, only 4-phenyl-2-butanol obviously lengthened the lag time. For the diphenolase activity, benzylideneacetone and benzylacetone were mixed-type inhibitors, while 4-phenyl-2-butanol was a noncompetitive type inhibitor. In conclusion, these compounds exhibited potent antityrosinase activities. This research would provide scientific evidence for the use of benzylideneacetone, benzylacetone, and 4-phenyl-2-butanol as antityrosinase agents.

Inhibitory effects of hinokitiol on tyrosinase activity and melanin biosynthesis and its antimicrobial activities.

The inhibitory effects of hinokitiol, a constituent of the woody oils isolated from Cupressaceae heartwood, on mushroom tyrosinase and melanin formation in B16 melanoma cells as well as its antimicrobial activity were investigated. Our results showed that hinokitiol could strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition was reversible. The IC(50) values were estimated as 9.67 µM for monophenolase activity and 0.21 µM for diphenolase activity. The lag time of the monophenolase activity was not obviously lengthened by the compound. Kinetic analyses showed that the inhibition mechanism of hinokitiol was a mixed-type inhibition of the diphenolase activity. Hinokitiol effectively inhibited both cellular tyrosinase activity and melanin biosynthesis in B16 melanoma cells with significant cytotoxicity. Furthermore, it was found that hinokitiol could inhibit the proliferation of Salmonella enteritidis, Escherichia coli, Bacillus subtilis, Staphyloccocus aureus, Klebsiella pneumoniae, and Ralstonia solanacearum to different extents. This research may widen the use of hinokitiol in the fields of food preservation, depigmentation, and insecticide use.

Inhibitory effects of 4-halobenzoic acids on the diphenolase and monophenolase activity of mushroom tyrosinase.

The effects of 4-halobenzoic acids (4-fluorobenzoic acid, 4-chlorobenzoic acid, and 4-bromobenzoic acid) on the activity of mushroom tyrosinase have been studied. The results show that 4-halobenzoic acids can strongly inhibit both monophenolase activity and diphenolase activity of the enzyme, and the inhibition displays a reversible course. The IC50 values were estimated as 0.26, 0.20, and 0.18 mM for diphenolase activity and as 1.03, 0.75, and 0.60 mM for monophenolase activity, respectively. Kinetic analyses show that the inhibition mechanism of all three 4-halobenzoic acids is noncompetitive inhibition to the diphenolase activity, and the inhibition constants (K1) were determined to be 0.25, 0.20, and 0.17 mM, respectively. The lag time of the monophenolase was obviously lengthened by these three 4-halobenzoic acids. When the concentration of inhibitors reached 1.4 mM, the lag time was lengthened from 30 s to 120, 125, and 150 s, respectively.