The mitochondrial genome sequence of the ghost crab, Ocypode ceratophthalmus, is documented (GenBank accession number: LN611669) in this article. This is the first mitogenome for the family Ocypodidae and the second for the order Ocypodoidea. Ocypode ceratophthalmus has a mitogenome of 15,564 base pairs consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the O. ceratophthalmus mitogenome is 35.78% for T, 19.36% for C, 33.73% for A and 11.13% for G, with an AT bias of 69.51% and the gene order is the typical arrangement for brachyuran crabs.
The Mictyris longicarpus (soldier crab) complete mitochondrial genome sequence is reported making it the first for the family Mictyridae and the second for the superfamily Ocypodoidea. The mitogenome is 15,548 base pairs made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The soldier crab mitogenome gene order is characteristic of brachyuran crabs with a base composition of 36.58% for T, 19.15% for C, 32.43% for A and 11.83% for G, with an AT bias of 69.01%.
Despite recent advances in sequencing technology, a complete mitogenome assembly is still unavailable for the gecarcinid land crabs that include the iconic Christmas Island red crab (Gecarcoidea natalis) which is known for its high population density, annual mass breeding migration and ecological significance in maintaining rainforest structure. Using sequences generated from Nanopore and Illumina platforms, we assembled the complete mitogenome for G. natalis, the first for the genus and only second for the family Gecarcinidae. Nine Nanopore long reads representing 0.15% of the sequencing output from an overnight MinION Nanopore run were aligned to the mitogenome. Two of them were >10 kb and combined are sufficient to span the entire G. natalis mitogenome. The use of Illumina genome skimming data only resulted in a fragmented assembly that can be attributed to low to zero sequencing coverage in multiple high AT-regions including the mitochondrial protein-coding genes (NAD4 and NAD5), 16S ribosomal rRNA and non-coding control region. Supplementing the mitogenome assembly with previously acquired transcriptome dataset containing high abundance of mitochondrial transcripts improved mitogenome sequence coverage and assembly reliability. We then inferred the phylogeny of the Eubrachyura using Maximum Likelihood and Bayesian approaches, confirming the phylogenetic placement of G. natalis within the family Gecarcinidae based on whole mitogenome alignment. Given the substantial impact of AT-content on mitogenome assembly and the value of complete mitogenomes in phylogenetic and comparative studies, we recommend that future mitogenome sequencing projects consider generating a modest amount of Nanopore long reads to facilitate the closing of problematic and fragmented mitogenome assemblies.
While the capacity for long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis has been elucidated in vertebrates and several invertebrate phyla, the comparative knowledge in crustaceans remains vague. A key obstacle in mapping the full spectrum of LC-PUFA biosynthesis in crustacean is the limited evidence of the functional activities of enzymes involved in desaturation or elongation of polyunsaturated fatty acid substrates. In this present study, we report on the cloning and functional characterization of two Elovl elongases from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis suggest these two Elovl as putative Elovl4 and Elovl6, respectively. Using the recombinant expression system in Saccharomyces cerevisiae, we demonstrate the elongation capacity for C18-C22 PUFA substrates in the S. olivacea Elovl4. The S. olivacea Elovl6 elongated saturated fatty acids, monounsaturated fatty acids, and interestingly, C18-C20 PUFA. Taken together, both Elovl fulfill the elongation steps required for conversion of C18 PUFA to their respective LC-PUFA products. Elovl4 is expressed mainly in the hepatopancreas and gill tissues, while Elovl6 is predominant in digestive tissues. The mRNA expression of both enzymes was higher in mud crabs fed with vegetable oil-based diets. Tissue fatty acid composition also showed the existence of LC-PUFA biosynthesis intermediate products in tissues expressing these two elongases. In summary, we report here two novel Elovl with PUFA elongating activities in a marine brachyuran. This will contribute significantly to the understanding of the LC-PUFA biosynthesis pathway in crustaceans and advance the development of aquafeed for intensive farming of the mud crab.
The mitochondrial genome sequence of the purple mottled shore crab, Cyclograpsus granulosus, is documented (GenBank accession number: LN624373), which makes it the third for genera of the superfamily Grapsoidea. Cyclograpsus granulosus has a mitogenome of 16,300 bp consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the C. granulosus mitogenome is 36.15% for T, 19.54% for C, 33.14% for A and 11.17% for G, with an AT bias of 69.29%. The mitogenome gene order is atypical for the brachyuran crabs, but is identical to species of the genus Eriocheir from the same family.
A primary factor in population management and wildlife conservation is the delineation of population units derived from descriptions of population genetic structure. Yet, predicting factors that influence the patterns of gene flow in a population particularly at landscape scales remains a major challenge in evolutionary biology. Here we report a population genetic study of the mud crab Scylla olivacea examined based on a 542 bp segment of the mitochondrial DNA cytochrome c oxidase I gene among 91 individuals from six localities in the west and east coast of Peninsular Malaysia. In total 55 unique haplotypes were distinguished with 45 private haplotypes and a single common haplotype shared among all populations studied. The other ten haplotypes were shared among various populations. The sharing of this haplotype reflects the connection of the mangrove areas between east and west coast of Peninsular Malaysia. High haplotype diversity (h = 0.968 ± 0.021; mean ± SD) and low nucleotide diversity (π = 0.120 ± 0.015; mean ± SD) were displayed, which may be indicative of genetic bottleneck events. No significant phylogenetic lineages were recognized using neighbour-joining and maximum parsimony methods. Hierarchical AMOVA analysis indicated that 99.33 % of the genetic variation was contained within populations and 0.67 % occurred among populations, suggesting no geographical patterning among populations studied, supported by F st test. Mismatch distribution analysis showed that the observed distribution of the pairwise mutation differences among haplotypes was multimodal, which is not concordant with a sudden range expansion scenario. However, neutrality tests showed non-significant negative values suggesting that the populations studied may have experienced past population growth, but the expansion may have been restricted to separate local areas that resulted in the non-significant negative Fu's Fs and Tajima's D value. Overall, this present preliminary study was able to be a reference on the phylogenetic relationships and assessment of genetic structure of Scylla sp. in Malaysia.
In this study, we first identified male-specific SNP markers using restriction site-associated DNA sequencing, and further developed a PCR-based sex identification technique for Charybdis feriatus. A total of 296.96 million clean reads were obtained, with 114.95 and 182.01 million from females and males. After assembly and alignment, 10 SNP markers were identified being heterozygous in males but homozygous in females. Five markers were further confirmed to be male-specific in a large number of individuals. Moreover, two male-specific sense primers and a common antisense primer were designed, using which, a PCR-based genetic sex identification method was successfully developed and used to identify the sex of 103 individuals, with a result of 49 females and 54 males. The presence of male-specific SNP markers suggests an XX/XY sex determination system for C. feriatus. These findings should be helpful for better understanding sex determination mechanism, and drafting artificial breeding program in crustaceans.
The molecular mechanism underlying sex determination and gonadal differentiation of the mud crab (Scylla paramamosain) has received considerable attention, due to the remarkably biological and economic differences between sexes. However, sex-biased genes, especially non-coding genes, which account for these differences, remain elusive in this crustacean species. In this study, the first de novo gonad transcriptome sequencing was performed to identify both differentially expressed genes and long non-coding RNAs (lncRNAs) between male and female S. paramamosain by using Illumina Hiseq2500. A total of 79,282,758 and 79,854,234 reads were generated from ovarian and testicular cDNA libraries, respectively. After filtrating and de novo assembly, 262,688 unigenes were produced from both libraries. Of these unigenes, 41,125 were annotated with known protein sequences in public databases. Homologous genes involved in sex determination and gonadal development pathways (Sxl-Tra/Tra-2-Dsx/Fru, Wnt4, thyroid hormone synthesis pathway, etc.) were identified. Three hundred and sixteen differentially expressed unigenes were further identified between both transcriptomes. Meanwhile, a total of 233,078 putative lncRNAs were predicted. Of these lncRNAs, 147 were differentially expressed between sexes. qRT-PCR results showed that nine lncRNAs negatively regulated the expression of eight genes, suggesting a potential role in sex differentiation. These findings will provide fundamental resources for further investigation on sex differentiation and regulatory mechanism in crustaceans.
Infection by the rhizocephalan parasite Sacculina beauforti can have detrimental effects on mud crab Scylla olivacea. However, the molecular changes that occur during rhizocephalan infection are poorly understood. Due to the disruption in the reproductive system after infection, the gonadal transcriptomic profiles of non-infected and infected Scylla olivacea were compared. A total of 686 and 843 unigenes were differentially expressed between non-infected and infected males, and females, respectively. The number of DEGs increased after infection. By comparing shared DEGs of non-infected and infected individuals, potential immune- and reproduction-related of host, and immune- and metabolism-related genes of parasite are highlighted. The only shared KEGG pathway between non-infected and infected individuals was the ribosome pathway. In summary, findings in this study provide new insights into the host-parasite relationship of rhizocephalan parasites and their crustacean hosts.
The infraorder Anomura consists of a morphologically and ecologically heterogeneous group of decapod crustaceans, and has attracted interest from taxonomists for decades attempting to find some order out of the seemingly chaotic diversity within the group. Species-level diversity within the Anomura runs the gamut from the "hairy" spindly-legged yeti crab found in deep-sea hydrothermal vent environments to the largest known terrestrial invertebrate, the robust coconut or robber crab. Owing to a well-developed capacity for parallel evolution, as evidenced by the occurrence of multiple independent carcinization events, Anomura has long tested the patience and skill of both taxonomists attempting to find order, and phylogeneticists trying to establish stable hypotheses of evolutionary inter-relationships. In this study, we performed genome skimming to recover the mitogenome sequences of 12 anomuran species including the world's largest extant invertebrate, the robber crab (Birgus latro), thereby over doubling these resources for this group, together with 8 new brachyuran mitogenomes. Maximum-likelihood (ML) and Bayesian-inferred (BI) phylogenetic reconstructions based on amino acid sequences from mitogenome protein-coding genes provided strong support for the monophyly of the Anomura and Brachyura and their sister relationship, consistent with previous studies. The majority of relationships within families were supported and were largely consistent with current taxonomic classifications, whereas many relationships at higher taxonomic levels were unresolved. Nevertheless, we have strong support for a polyphyletic Paguroidea and recovered a well-supported clade of a subset of paguroids (Diogenidae + Coenobitidae) basal to all other anomurans, though this requires further testing with greater taxonomic sampling. We also introduce a new feature to the MitoPhAST bioinformatics pipeline (https://github.com/mht85/MitoPhAST) that enables the extraction of mitochondrial gene order (MGO) information directly from GenBank files and clusters groups based on common MGOs. Using this tool, we compared MGOs across the Anomura and Brachyura, identifying Anomura as a taxonomic "hot spot" with high variability in MGOs among congeneric species from multiple families while noting the broad association of highly-rearranged MGOs with several anomuran lineages inhabiting extreme niches. We also demonstrate the value of MGOs as a source of novel synapomorphies for independently reinforcing tree-based relationships and for shedding light on relationships among challenging groups such as the Aegloidea and Lomisoidea that were unresolved in phylogenetic reconstructions. Overall, this study contributes a substantial amount of new genetic material for Anomura and attempts to further resolve anomuran evolutionary relationships where possible based on a combination of sequence and MGO information. The new feature in MitoPhAST adds to the relatively limited number of bioinformatics tools available for MGO analyses, which can be utilized widely across animal groups.
The crucifix crab, Charybdis feriatus, which mainly inhabits Indo-Pacific region, is regarded as one of the most high-potential species for domestication and incorporation into the aquaculture sector. However, the regulatory mechanisms of sex determination and differentiation of this species remain unclear. To identify candidate genes involved in sex determination and differentiation, high throughput sequencing of transcriptome from the testis and ovary of C. feriatus was performed by the Illumina platform. After removing adaptor primers, low-quality sequences and very short (<50 nt) reads, we obtained 80.9 million and 66.2 million clean reads from testis and ovary, respectively. A total of 86,433 unigenes were assembled, and ~43% (37,500 unigenes) were successfully annotated to the NR, NT, Swiss-Prot, KEGG, COG, GO databases. By comparing the testis and ovary libraries, we obtained 27,636 differentially expressed genes. Some candidate genes involved in the sex determination and differentiation of C. feriatus were identified, such as vasa, pgds, vgr, hsp90, dsx-f, fem-1, and gpr. In addition, 88,608 simple sequence repeats were obtained, and 61,929 and 77,473 single nucleotide polymorphisms from testis and ovary were detected, respectively. The transcriptome profiling was validated by quantitative real-time PCR in 30 selected genes, which showed a good consistency. The present study is the first high-throughput transcriptome sequencing of C. feriatus. These findings will be useful for future functional analysis of sex-associated genes and molecular marker-assisted selections in C. feriatus.
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.
Adequate genetic information is essential for sustainable crustacean fisheries and aquaculture management. The commercially important orange mud crab, Scylla olivacea, is prevalent in Southeast Asia region and is highly sought after. Although it is a suitable aquaculture candidate, full domestication of this species is hampered by the lack of knowledge about the sexual maturation process and the molecular mechanisms behind it, especially in males. To date, data on its whole genome is yet to be reported for S. olivacea. The available transcriptome data published previously on this species focus primarily on females and the role of central nervous system in reproductive development. De novo transcriptome sequencing for the testes of S. olivacea from immature, maturing and mature stages were performed. A total of approximately 144 million high-quality reads were generated and de novo assembled into 160,569 transcripts with a total length of 142.2 Mb. Approximately 15-23% of the total assembled transcripts were annotated when compared to public protein sequence databases (i.e. UniProt database, Interpro database, Pfam database and Drosophila melanogaster protein database), and GO-categorised with GO Ontology terms. A total of 156,181 high-quality Single-Nucleotide Polymorphisms (SNPs) were mined from the transcriptome data of present study. Transcriptome comparison among the testes of different maturation stages revealed one gene (beta crystallin like gene) with the most significant differential expression-up-regulated in immature stage and down-regulated in maturing and mature stages. This was further validated by qRT-PCR. In conclusion, a comprehensive transcriptome of the testis of orange mud crabs from different maturation stages were obtained. This report provides an invaluable resource for enhancing our understanding of this species' genome structure and biology, as expressed and controlled by their gonads.