De novo assembly of the transcriptome of Acanthaster plancitestes

A key strategy to reduce coral loss is the development of effective control method for the corallivorous crown-of-thorns sea star (Acanthaster planci), an omnipresent scourge and threat to the biodiversity of reefs in the Indo-Pacific region. Limited genetic resources are available for this highly fecund species. In this study, we explored one aspect at the heart of A. planci outbreaks, the male reproductive system. Using high-throughput sequencing technology, we report for first time the production of a comprehensive transcriptomic data set for the testes of A. placni that can aid in understanding the molecular mechanisms involved in A. planci spermatogenesis and fertilization. Through de novo transcriptome sequencing, we produced 52 965 998 raw reads corresponding to 4.76 Gb clean read data. From this, 243 870 contigs were assembled with Trinity and used to construct 92 792 unigenes. Distinct genes were then annotated with blastx yielding 30 810 unigenes above the cut-off E-value set at 10−5, with ESTScan database query analyses yielding up to 5366 unigenes to known hits. The identification of genes directly involved in sperm development (DEAD-box family proteins), motility, fertilization and signalling (Bindin/Speract receptor) are also discussed

Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for histones in mature sperm nuclei

Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1&ndash;3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.<br /

The presence of GABA in gastropod mucus and its role in inducing larval settlement

Chemical substances that induce larval settlement have been the focus of many gastropod studies due to the importance of wild stock recruitment and production within aquaculture facilities. Gamma-aminobutyric acid (GABA), GABA analogs, and GABA-mimetics associated with certain crustose coralline algae (CCA), are known to induce larval settlement in commercial abalone (Haliotis) species, and other gastropods. Furthermore, mucus secreted from these gastropods has been shown to induce larval settlement, but the stimulatory components of mucus have not been thoroughly investigated. We now present data confirming that GABA is the settlement-inducing effector molecule contained within abalone mucus. To do this, we initially generated anti-GABA for use in immunoenzyme and immunofluorescent microscopy. Using these techniques GABA was identified in the nerves and epithelial cells of the foot, including mucus. Dried mucus samples subject to HPLC analysis revealed a mean concentration of 0.68 mM GABA after sample rehydration. The presence of GABA in these samples was confirmed by time-of-flight mass spectroscopy (TOF-MS). In addition, GABA was detected in the mucus of several abalone species and other gastropods by immunocytochemistry. Subsequent bioassays using both dry and fresh mucus strongly promoted induction of larval settlement.<br /

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ-sinus gland (XO-SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHHtype I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XOSG, raises new questions concerning the established mode of MIH action

Characterization of a GABA(A) receptor beta subunit in the abalone Haliotis asinina that is upregulated during larval development

In the tropical abalone Haliotis asinina, the neurotransmitter gamma-aminobutyric acid (GABA) is a potent inducer of larval settlement, a process beginning with the onset of a behavioral search for a suitable substratum and ending with metamorphosis. In the natural environment, larvae can encounter GABA or GABA-like molecules through association with conspecific foot mucus and crustose coralline algae. To understand the role of GABA in the molecular process leading to settlement required identification and analysis of GABA's cognate receptor. We now have isolated the first abalone full-length GABA(A) receptor (Has-GABA(A)R) beta subunit gene, which encodes a protein of 485 amino acids, from juvenile H. asinina neural tissue. Similar to other metazoan GABA(A)Rs, the abalone GABA(A)R contains four transmembrane domains, a conserved cysteine loop in the N-terminal extra-cellular domain, and highly conserved sequence motifs. The Has-GABA(A)R gene is expressed at extremely low levels in unfertilized eggs, but increases significantly just prior to settlement, peaking at 120 h post fertilization (hpf). We further demonstrate that during the period of larval competence (96-144 hpf), gene transcripts and the encoded Has-GABA(A)R were localized in a cluster of cells along the dorsal and lateral edges of the foot, as well as the posterior epithelium. In functional settlement assays using GABA and 5-AVA, we found that there was significantly lower settlement of veligers pre-treated with antibodies to an external domain of the Has-GABA(A)R than those treated with preimmune serum, or untreated veligers. We postulate that this receptor may act as a highly sensitive chemical sensor, whose activation is necessary to trigger chloride-mediated sensory neuron activation or inhibition, leading to the initiation of settlement and metamorphosis events. (C) 2011 Elsevier B.V. All rights reserved

Identification of Gene Biomarkers for Tigilanol Tiglate Content in Fontainea picrosperma

Tigilanol tiglate (EBC-46) is a small-molecule natural product under development for the treatment of cancers in humans and companion animals. The drug is currently produced by purification from the Australian rainforest tree Fontainea picrosperma (Euphorbiaceae). As part of a selective-breeding program to increase EBC-46 yield from F. picrosperma plantations, we investigated potential gene biomarkers associated with biosynthesis of EBC-46. Initially, we identified individual plants that were either high (&gt;0.039%) or low EBC-46 (&lt;0.008%) producers, then assessed their differentially expressed genes within the leaves and roots of these two groups by quantitative RNA sequencing. Compared to low EBC-46 producers, high-EBC-46-producing plants were found to have 145 upregulated genes and 101 downregulated genes in leaves and 53 upregulated genes and 82 downregulated genes in roots. Most of these genes were functionally associated with defence, transport, and biosynthesis. Genes identified as expressed exclusively in either the high or low EBC-46-producing plants were further validated by quantitative PCR, showing that cytochrome P450 94C1 in leaves and early response dehydration 7.1 and 2-alkenal reductase in roots were consistently and significantly upregulated in high-EBC-46 producers. In summary, this study has identified biomarker genes that may be used in the selective breeding of F. picrosperma

Distribution of Gaba in the nerve ganglia of haliotis asinina linnaeus

Gamma-aminobutyric acid (GABA) is a major neurotransmitter and effective settlement inducer in abalone aquaculture. This study aimed to explore the distribution of GABA within neural tissues of Haliotis asinina. Gamma-aminobutyric acid was found in neuronal cell type 1 of 3 major ganglia (i.e., cerebral, pleuropedal, and visceral ganglia) of both sexes. The distribution of GABA-immunoreactive (-ir) cells in the cerebral ganglion was concentrated mostly in the cortex region of the dorsal horn, whereas it was scattered throughout the pleuropedal ganglion, with more in the upper half. Gamma-aminobutyric acid-ir nerve fibers were found throughout the neuropils of the ganglia. The visceral ganglion had the least numbers of GABA-ir neurons compared with the other 2 ganglia. The cells were distributed mainly in the dorsal horn. We also observed GABA to be colocalized with 2 other neurotransmitters: serotonin (5-HT) and dopamine (DA). In the cerebral ganglion, fluorescence double staining of GABA and 5-HT, and GABA and DA showed immunoreactivity in separate cells and was also colocalized in the same cells. In the pleuropedal ganglion, the staining pattern was similar to the cerebral ganglion, but positive-staining cells were less numerous. In the visceral ganglion, GABA and DA, and GABA and 5-HT were colocalized in the same cell types. Overall, we found that GABAergic cells were most numerous in the cerebral ganglion of H. asinina. Further studies are required to determine the functions of these neurotransmitters in relation to their distribution