Additional file 1 of Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection

Supplementary Material

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

DataSheet_1_Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon.pdf

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation.</p

Table_2_Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon.xlsx

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation.</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

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

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

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

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