Hanafiah Fazhan

Transcriptome Analysis and Differential Gene Expression on the Testis of Orange Mud Crab, Scylla olivacea, during Sexual Maturation

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.

Use of Abdomen Looseness as an Indicator of Sexual Maturity in Male Mud Crab Scylla spp.

ABSTRACT Stock assessment in wild populations and research involving reproductive biology of crabs rely greatly on the ability to accurately determine sexual maturity status. Although determination of sexual maturity in male mud crabs was commonly based on the presence of spermatophores in the vas deferens, preliminary studies on the mud crab Scylla spp. showed that this method is inconclusive and was reevaluated in this study. In addition, the feasibility of using male abdomen looseness as maturity indicator was explored. Spermatophores were present in the vas deferens of all Scylla spp. regardless of maturity status and body size. Spermatophores also significantly increased in size as the crabs reached sexual maturity. Examination of the vas deferens external morphology (e.g., color and size) consistently differentiated immature from mature males. The size at maturity (CW50) was successfully estimated using three maturity indicators, that is, the looseness of the abdomen, the observation of mating scars, and the vas deferens development. The estimated sizes obtained using all three indicators showed no significant differences, but varied significantly among species. A positive relationship was observed between body size and sexual maturity in all three Scylla species. Determination of sexual maturity using the presence of mating scars as indicator was inconsistent as some crabs that were mature in terms of looseness of abdomen and vas deferens development showed no presence of mating scars. On the other hand, male crabs with loose abdomens were found to be sexually matured with enlarged, milky white vas deferens. This finding suggests that abdomen looseness is an accurate sexual maturity indicator in males. Using looseness of abdomen as sexual maturity indicator in the male Scylla spp., therefore, is feasible and superior to conventional methods, with the advantages of being practical, easy to carry out, and does not require sacrificing of the crabs.

The complete mitochondrial genome of the swimming crab Charybdis natator (Herbst) (Decapoda: Brachyura: Portunidae) and its phylogeny

Abstract The complete mitochondrial genome of Charybdis natator (family Portunidae) was obtained using Illumina and Sanger dideoxy sequencing. This genome was a typically circular molecule with a length of 15,664 bp and encoded 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The overall base composition of this mitogenome was 34.00% for A, 11.06% for G, 36.36% for T, and 18.58% for C, respectively, with a higher A + T content (70.36%). The gene composition and arrangement were accordant to the closely published species. The phylogenetic analysis suggested that C. natator had the closest relationship with C. japonica.

The complete mitochondrial genome and phylogenetic analysis of Matuta planipes (Decapoda: Brachyura: Matutidae)

Abstract The complete mitochondrial genome of Matuta planipes was obtained using long and conventional PCR method. The circular genome was 15,760 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a control region. Of the 37 genes, 23 were encoded by the heavy strand, while the others were encoded by the light strand. The genome composition with A + T bias (70.82%) and gene arrangement were largely identical to those observed in most arthropods, such as the mud crab (Scylla paramamosain). The phylogenetic analysis suggested that M. planipes was closest to Ashtoret lunaris. The newly described mitochondrial genome may provide valuable data for phylogenetic analysis for Matutidae.

Infestation of parasitic rhizocephalan barnacles Sacculina beauforti (Cirripedia, Rhizocephala) in edible mud crab, Scylla olivacea

Screening of mud crab genus Scylla was conducted in four locations (Marudu Bay, Lundu, Taiping, Setiu) representing Malaysia. Scylla olivacea with abnormal primary and secondary sexual characters were prevalent (approximately 42.27% of the local screened S. olivacea population) in Marudu Bay, Sabah. A total of six different types of abnormalities were described. Crabs with type 1 and type 3 were immature males, type 2 and type 4 were mature males, type 5 were immature females and type 6 were mature females. The abdomen of all crabs with abnormalities were dented on both sides along the abdomen’s middle line. Abnormal crabs showed significant variation in their size, weight, abdomen width and/or gonopod or pleopod length compared to normal individuals. The mean body weight of abnormal crabs (type 1–5) were higher than normal crabs with smaller body size, while females with type 6 abnormality were always heavier than the normal counterparts at any given size. Sacculinid’s externa were observed in the abdomen of crabs with type 4 and type 6 abnormalities. The presence of embryos within the externa and subsequent molecular analysis of partial mitochondrial COI region confirmed the rhizocephalan parasite as Sacculina beauforti. Future in-depth descriptions of the life cycle and characteristics of S. beauforti are recommended as it involves a commercially important edible crab species and the effect on human health from the consumption of crabs is of crucial concern.

Transcriptome-seq provides insights into sex-preference pattern of gene expression between testis and ovary of the crucifix crab (Charybdis feriatus)

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 whole mitochondrial genome and phylogenetic analysis of Lupocycloporus gracilimanus (Stimpson, 1858) (Decapoda, portunidae)

Abstract The mitochondrial genome plays an important role in studies on phylogeography and population genetic diversity. Here we report the complete mitochondrial genome of Lupocycloporus gracilimanus (Stimpson, 1858) which is the first mitochondrial genome reported in genus Lupocycloporus by now. The mitogenome is 15,990 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a putative control region. The phylogenetic analysis showed that L. gracilimanus was closest to genus Scylla. The present research should provide valuable information for phylogenetic analysis and classification of Portunidae.

Identification of the complete mitochondrial genome of Monomia gladiator (Decapoda: Brachyura: Portunidae) and its phylogenetic relationship

Abstract The complete mitochondrial genome sequence plays an important role in phylogenetic studies. In this study, the complete mitochondrial genome of Monomia gladiator was obtained by Illumina and Sanger sequencing techniques. The circular genome was 15,878 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a putative control region. This whole mitogenome composition was 33.32% for A, 35.69% for T, 11.75% for G, and 19.24% for C, respectively. The phylogenetic analysis suggested that M. gladiator was genetically closest to three Portunidae species (Charybdis japonica, C. feriata and Thalamita crenata). The newly described mitogenome may facilitate the phylogenetic studies on Portunidae crabs.

Characterization of the complete mitochondrial genome of Xanthid crab, Atergatis integerrimus from China (Decapoda: Brachyura) and its phylogenetic analysis

Abstract The complete mitochondrial genome sequence of Atergatis integerrimus from China has been amplified and sequenced in this study. The mitogenome assembly was found to be 15,924 bp in length with base composition of A (32.88%), G (10.58%), C (20.87%), T (35.66%), A + T (68.54%), and G + C (31.46%). It contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a control region. The phylogenetic position was constructed and the A. integerrimus was closely clustered with Pseudocarcinus gigas and Leptodius sanguineus. The complete mitochondrial genome sequence would be useful for further understanding the evolution of A. integerrimus.

The complete mitochondrial genome of Varuna yui (Decapoda: Brachyura: Varunidae) and its phylogeny

Abstract The complete mitochondrial genome plays an important role in the research on phylogenetic relationship. Here, we reported the first complete mitochondrial genome sequence of Varuna yui Hwang & Takeda, 1986 (Varunidae). The complete mtDNA (15,915 bp in length) consisted of 13 protein-coding genes, 22 tRNAs, two rRNA genes, and a control region. The gene arrangement was identical to those observed in the Varunidae species. The phylogenetic analysis suggested that V. yui had close relationship with other Varunidae species (Helicetient sinensis, Eriocher sinesis, etc.). The newly described genome may facilitate further comparative mitogenomic analysis within Varunidae species.