Effect of priming 5-HT on the relative <i>PGFS</i> gene expression levels of <i>S. olivacea</i> using quantitative RT-PCR.

<p><i>PGFS</i> mRNA expression levels in the brain, VNC and ovary (n = 12) were determined at 3, 6, 12 and 24 h post 5-HT injection. Data were normalized against <i>β</i>-actin. Asterisks indicate significant differences (<i>p<</i>0.05) with respect to the control (vehicle-injected) group. Abbreviations: prostaglandin F synthase (PGFS), ventral nerve cord (VNC), ovary (OV), and vehicle control (VC).</p

Alignment of amino acid sequences of farnesoic acid o-methyltransferase (<i>FAMeT</i>) of <i>S. olivacea</i> with known arthropod species.

<p>Gaps (-) included to allow for alignment. “*”: identical amino acids; “:”: conserved substitutions (same group); “.”: semi-conserved substitution (similar shapes). Circles indicating phosphorylation site prediction on protein sequences. (<i>Scyol</i>, <i>Scylla olivacea</i>) orange mud crab; (<i>Scypa</i>, <i>Scylla paramamosain</i>), green mud crab (Accession number ADK32330); (<i>Canpa</i>, <i>Cancer pagurus</i>), edible crab (AAR00732); (<i>Meten</i>, <i>Metapenaeus ensis</i>), sand shrimp (AAK28535); (<i>Penmo</i>, <i>Penaeus monodon</i>), black tiger shrimp (AAX24112); (<i>Macni</i>, <i>Macrobrachium nipponense</i>), freshwater prawn (AFA26604); and (<i>Pappo</i>, <i>Papilio polytes</i>), butterfly (BAM20323).</p

List of putative neuropeptides, predicted from non-hit blast search transcripts.

<p><b>**Based on precursor signal sequence (SignalP 4.1).</b></p><p>List of putative neuropeptides, predicted from non-hit blast search transcripts.</p

Identification of Genes Associated with Reproduction in the Mud Crab (<i>Scylla olivacea</i>) and Their Differential Expression following Serotonin Stimulation

<div><p>The central nervous system (CNS) is often intimately involved in reproduction control and is therefore a target organ for transcriptomic investigations to identify reproduction-associated genes. In this study, 454 transcriptome sequencing was performed on pooled brain and ventral nerve cord of the female mud crab (<i>Scylla olivacea</i>) following serotonin injection (5 µg/g BW). A total of 197,468 sequence reads was obtained with an average length of 828 bp. Approximately 38.7% of 2,183 isotigs matched with significant similarity (E value < 1e⁻⁴) to sequences within the Genbank non-redundant (nr) database, with most significant matches being to crustacean and insect sequences. Approximately 32 putative neuropeptide genes were identified from nonmatching blast sequences. In addition, we identified full-length transcripts for crustacean reproductive-related genes, namely farnesoic acid o-methyltransferase (<i>FAMeT</i>), estrogen sulfotransferase (<i>ESULT</i>) and prostaglandin F synthase (<i>PGFS</i>). Following serotonin injection, which would normally initiate reproductive processes, we found up-regulation of <i>FAMeT</i>, <i>ESULT</i> and <i>PGFS</i> expression in the female CNS and ovary. Our data here provides an invaluable new resource for understanding the molecular role of the CNS on reproduction in <i>S. olivacea</i>.</p></div

DataSheet_1_Acceleration of Ovarian Maturation in the Female Mud Crab With RNA Interference of the Vitellogenesis-Inhibiting Hormone (VIH).pdf

In the present study, double strand RNA technology (dsRNA) was used to inhibit transcripts of vitellogenesis-inhibiting hormone (VIH) that mainly synthesized and secreted from the central nervous system in Scylla olivacea females. The results presented in this study clearly demonstrate the potential dsRNA-VIH was highly effective to inhibit VIH in the eyestalks of females injected with dsRNA-VIH on the 3rd, 7th and 14th day, respectively. The dsRNA-VIH injections were performed at 14-day intervals, a single dsRNA dose of 0.6 µg/gram body weight was enough to suppress VIH expression until 14th day after injection. The dsRNA-VIH injection significantly increased gonad-somatic index (GSI) and hemolymph vitellin level at day 14 and 28 when compared with control groups. The histological observation found that the number of oocyte step 4 in dsRNA-VIH group was significantly higher than that of the control group. Also, dsRNA-VIH has stimulatory function on other reproduction-related genes such as the Scyol-PGES and Scyol-ESULT that both genes gradually increased their expressions in brain and ventral nerve cord. In conclusion, the silence of VIH gene could reduce the production of VIH from eyestalk and brain that affected other downstream genes related to ovarian maturation in the mud crab.</p

Gene ontology (GO) annotations (level 2 terms) for the transcriptomic sequences of <i>S. olivacea</i>.

<p>(A) biological process, (B) molecular function, and (C) cellular component.</p

Alignment of amino acid sequence of prostaglandin F synthase (<i>PGFS</i>) of <i>S. olivacea</i> with known sequences from other species.

<p>Gaps (-) included to allow alignment. “*”: identical amino acids; “:”: conserved substitutions (same group); “.”: semi-conserved substitution (similar shapes). Circles indicating phosphorylation site prediction on protein sequences. (<i>Scyol</i>, <i>Scylla olivacea</i>), mud crab; (<i>Otoga</i>, <i>Otolemur garnettii</i>), greter galago (Accession number XP_003793364); (<i>Musmu</i>, <i>Mus musculus</i>) Mouse (NP_079858); (<i>Panpa</i>, <i>Pan paniscus</i>), bonbono (XP_003807368); (<i>Brafl</i>, <i>Branchiostoma floridae</i>), lancelet (XP_002587381); (<i>Ixori</i>, <i>Ixodes ricinus</i>), castor bean tick (JAA72765); and (<i>Dappu</i>, <i>Daphnia pulex</i>), water flea (EFX68017).</p

Effect of priming 5-HT on relative <i>FAMeT</i> gene expression level in <i>S. olivacea</i> using quantitative RT-PCR.

<p><i>FAMeT</i> mRNA expression levels in the brain, VNC and ovary (n = 12) were determined at 3, 6, 12 and 24 h post 5-HT injection. Data were normalized against <i>β</i>-actin. Asterisks indicate significant differences (<i>p<</i>0.05) with respect to the control (vehicle-injected) group. Abbreviations: farnesoic acid o-methyltransferase (FAMeT), ventral nerve cord (VNC), ovary (OV), and vehicle control (VC).</p

Blastx top hits (30 species) using <i>S. olivacea</i> CNS isotigs (E-value cut-off 1e⁻⁴).

<p>Distribution of known species is primarily crustacean and other arthropods.</p

Specific primers used for RACE PCR and real time quantitative RT-PCR.

<p>Specific primers used for RACE PCR and real time quantitative RT-PCR.</p