Springer Science and Business Media LLC

We investigated microorganisms associated with a deep-sea sponge, Characella sp. (Pachastrellidae) collected at a hydrothermal vent site (686 m depth) in the Sumisu Caldera, Ogasawara Island chain, Japan, and with two sponges, Pachastrella sp. (Pachastrellidae) and an unidentified Poecilosclerida sponge, collected at an oil seep (572 m depth) in the Gulf of Mexico, using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) directed at bacterial 16S rRNA gene sequences. In the PCR-DGGE profiles, we detected a single clearly dominant band in each of the Characella sp. and the unidentified Poecilosclerida sponge. BLAST search of their sequences showed that they were most similar (>99% identity) to those of the gammaproteobacterial thioautotrophic symbionts of deep-sea bivalves from hydrothermal vents, Bathymodiolus spp. Phylogenetic analysis of the near-full length sequences of the 16S rRNA genes cloned from the unidentified Poecilosclerida sponge and Characella sp. confirmed that they were closely related to thioautotrophic symbionts. Although associations between sponges and methanotrophic bacteria have been reported previously, this is the first report of a possible stable association between sponges and thioautotrophic bacteria.

In this study, we trialed 6-dimethylaminopurine (6-DMAP) chemical shocks to induce meiosis I or meiosis II Pacific White shrimp, Litopenaeus vannamei, triploids for the first time, and cold temperature shocks to induce meiosis II L. vannamei triploids as done previously. Inductions were performed on 37 spawnings in total with experiments being progressively designed in a factorial manner to allow optimization of induction parameters. Treatment with a 200-μm 6-DMAP final concentration at 1 min post-spawning detection for a 6 to 8 min duration resulted in the most consistent induction of chemically induced meiosis I triploids while treatment at 7 min 30 s post-spawning detection for a 10-min duration resulted in the most consistent induction of chemically induced meiosis II triploids. A cold temperature shock of 11.7°C to 13.25°C (final treatment temperature; spawning water temperature 28.5°C) applied at 8 min post-spawning detection for a 4 to 10 min duration resulted in the most consistent induction of cold-temperature-induced meiosis II triploids. 6-DMAP shocks resulted in meiosis I induction rates from 29% to 100% in unhatched embryos and 50% in nauplii, and meiosis II induction rates from 65% to 100% in unhatched embryos and 52% to 100% in nauplii. Cold shocks resulted in induction rates from 5% to 100% in unhatched embryos and nauplii. Confocal microscopy analysis of embryos revealed that there are major developmental abnormalities in a large proportion of later stage triploid L. vannamei embryos compared to their diploid sibling controls. Despite this, however, some triploid embryos did appear normal and both shock agents induced small numbers of viable triploid L. vannamei nauplii which were successfully reared to protozoeal stage 3 as confirmed by flow cytometry. Triploids beyond this life-history stage were not observed in the present study as confirmed by flow cytometry at mysis stages. This study adds to our knowledge base of triploid induction in L. vannamei and further highlights the inherent difficulties with triploid embryonic and larval viability in this species.

Investigations of host-symbiont interactions can benefit enormously from a complete and reliable holobiont gene expression profiling. The most efficient way to acquire holobiont transcriptomes is to perform RNA-Seq on both host and symbionts simultaneously. However, optimal methods for capturing both host and symbiont mRNAs are still under development, particularly when the host is a eukaryote and the symbionts are bacteria or archaea. Traditionally, poly(A)-enriched libraries have been used to capture eukaryotic mRNA, but the ability of this method to adequately capture bacterial mRNAs is unclear because of the short half-life of the bacterial transcripts. Here, we address this gap in knowledge with the aim of helping others to choose an appropriate RNA-Seq approach for analysis of animal host-bacterial symbiont transcriptomes. Specifically, we compared transcriptome bias, depth and coverage achieved by two different mRNA capture and sequencing strategies applied to the marine demosponge Amphimedon queenslandica holobiont. Annotated genomes of the sponge host and the three most abundant bacterial symbionts, which can comprise up to 95% of the adult microbiome, are available. Importantly, this allows for transcriptomes to be accurately mapped to these genomes, and thus quantitatively assessed and compared. The two strategies that we compare here are (i) poly(A) captured mRNA-Seq (Poly(A)-RNA-Seq) and (ii) ribosomal RNA depleted RNA-Seq (rRNA-depleted-RNA-Seq). For the host sponge, we find no significant difference in transcriptomes generated by the two different mRNA capture methods. However, for the symbiont transcriptomes, we confirm the expectation that the rRNA-depleted-RNA-Seq performs much better than the Poly(A)-RNA-Seq. This comparison demonstrates that RNA-Seq by ribosomal RNA depletion is an effective and reliable method to simultaneously capture gene expression in host and symbionts and thus to analyse holobiont transcriptomes.

The striped bass (Morone saxatilis) and its relatives (genus Morone) are of great importance to fisheries and aquaculture in North America. As part of a collaborative effort to employ molecular genetics technologies in striped bass breeding programs, we previously developed nearly 500 microsatellite markers. The objectives of this study were to construct a microsatellite linkage map of striped bass and to examine conserved synteny between striped bass and three-spined stickleback (Gasterosteus aculeatus). Of 480 microsatellite markers screened for polymorphism, 289 informative markers were identified and used to genotype two half-sib mapping families. Twenty-six linkage groups were assembled, and only two markers remain unlinked. The sex-averaged map spans 1,623.8 cM with an average marker density of 5.78 cM per marker. Among 287 striped bass microsatellite markers assigned to linkage groups, 169 (58.9%) showed homology to sequences on stickleback chromosomes or scaffolds. Comparison between the stickleback genome and the striped bass linkage map revealed conserved synteny between these two species. This is the first linkage map for any of the Morone species. This map will be useful for molecular mapping and marker-assisted selection of genes of interest in striped bass breeding programs. The conserved synteny between striped bass and stickleback will facilitate fine mapping of genome regions of interest and will serve as a new resource for comparative mapping with other Perciform fishes such as European sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata), and tilapia (Oreochromis ssp.).

Fish are the main source of the n-3 highly unsaturated fatty acids, which are crucial for human health. Their synthesis from C18 precursors is mediated by desaturases and elongases, but the activity of these enzymes has not been conclusively established in marine fish species. This study reports the cloning, tissue expression, and functional characterization of a sea bass (Dicentrarchus labrax L.) Δ6-desaturase and one of its splicing variants. Two cDNAs with open reading frames of 1,346 and 1,354 bp were cloned and named D6D and D6D-V, respectively. Both deduced protein sequences (445 and 387 amino acids, respectively) contained two transmembrane regions and the N-terminal cytochrome b5 domain with the HPGG motif characteristic of microsomal desaturases. D6D presents three histidine-rich regions, whereas in D6D-V, an insertion of eight nucleotides in the boundaries of exons 10 and 11 modified the third histidine-rich domain and led to insertion of a premature STOP codon, resulting in a shorter predicted protein. Quantitative real-time polymerase chain reaction assay of gene expression showed that D6D was highly expressed in the brain and intestine, and to a lesser extent, in muscle and liver; meanwhile, D6D-V was expressed in all tissues tested, but at level at least 200-fold lower than D6D. Functional analysis in yeast showed that sea bass D6D encodes a fully functional Δ6-desaturase with no residual Δ5-desaturase activity. This desaturase does not exhibit a clear preference for n-3 versus n-6 C18 substrates. Interestingly, D6D-V is a nonfunctional protein, suggesting that the C-terminal end is indispensable for protein activity.