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

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

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

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

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

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

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

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

Alignment of <i>S. olivacea</i> amino acid sequence of estrogen sulfotransferase (<i>ESULT</i>) with similar sequence from other 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>Trica</i>, <i>Tribolium castaneum</i>), beetle (Accession number EFA11828); (<i>Dappu</i>, <i>Daphnia pulex</i>), water flea (EFX82151.1); (<i>Megro</i>, <i>Megachile rotundata</i>), leafcutter bee (XP_003706084); and (<i>Bomim</i>, <i>Bombus impatiens</i>), bumble bee (XP_003485365).</p