Infection by the Parasite Myxobolus bejeranoi (Cnidaria: Myxozoa) Suppresses the Immune System of Hybrid Tilapia.

Myxozoa (Cnidaria) is a large group of microscopic obligate endoparasites that can cause emerging diseases, affecting wild fish populations and fisheries. Recently, the myxozoan Myxobolus bejeranoi was found to infect the gills of hybrid tilapia (Nile tilapia (Oreochromis niloticus) × Jordan/blue tilapia (O. aureus)), causing high morbidity and mortality. Here, we used comparative transcriptomics to elucidate the molecular processes occurring in the fish host following infection by M. bejeranoi. Fish were exposed to pond water containing actinospores for 24 h and the effects of minor, intermediate, and severe infections on the sporulation site, the gills, and on the hematopoietic organs, head kidney and spleen, were compared. Enrichment analysis for GO and KEGG pathways indicated immune system activation in gills at severe infection, whereas in the head kidney a broad immune suppression included deactivation of cytokines and GATA3 transcription factor responsible for T helper cell differentiation. In the spleen, the cytotoxic effector proteins perforin and granzyme B were downregulated and insulin, which may function as an immunomodulatory hormone inducing systemic immune suppression, was upregulated. These findings suggest that M. bejeranoi is a highly efficient parasite that disables the defense mechanisms of its fish host hybrid tilapia.

Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis.

The metabotropic gamma-aminobutyric acid B receptor (GABABR) is a G protein-coupled receptor that mediates neuronal inhibition by the neurotransmitter GABA. While GABABR-mediated signalling has been suggested to play central roles in neuronal differentiation and proliferation across evolution, it has mostly been studied in the mammalian brain. Here, we demonstrate that ectopic activation of GABABR signalling affects neurogenic functions in the sea anemone Nematostella vectensis. We identified four putative Nematostella GABABR homologues presenting conserved three-dimensional extracellular domains and residues needed for binding GABA and the GABABR agonist baclofen. Moreover, sustained activation of GABABR signalling reversibly arrests the critical metamorphosis transition from planktonic larva to sessile polyp life stage. To understand the processes that underlie the developmental arrest, we combined transcriptomic and spatial analyses of control and baclofen-treated larvae. Our findings reveal that the cnidarian neurogenic programme is arrested following the addition of baclofen to developing larvae. Specifically, neuron development and neurite extension were inhibited, resulting in an underdeveloped and less organized nervous system and downregulation of proneural factors including NvSoxB(2), NvNeuroD1 and NvElav1. Our results thus point to an evolutionarily conserved function of GABABR in neurogenesis regulation and shed light on early cnidarian development.

Morphological and molecular characterization of a novel myxosporean parasite Myxobolus bejeranoi n. sp. (Cnidaria: Myxosporea) from hybrid tilapia in Israel.

Myxosporean infections can cause severe damage to commercially grown tilapia. Here, we report a novel myxosporean that was found in gills of Oreochromis aureus male × Oreochromis niloticus female, which is an important aquaculture tilapia hybrid in Israel. Three-month-old fish were found to have cysts located in gill muscle tissue, which were filled with both immature and mature spores. Affected fish displayed higher mortality rate. Spore dimensions (10.8 ± 0.7 µm length × 6.8 ± 0.6 µm width) and molecular characterization using 18S ribosomal DNA revealed that the unknown parasite belongs in the Myxobolus clade. Based on the infection site, spore morphology and molecular characterization, we describe this parasite as Myxobolus bejeranoi n. sp. (MF401455). Phylogenetic analysis showed that the new species is most closely related to two Myxobolus spp. from O. niloticus in Egypt and Ghana.

Transcriptome profiling of the dynamic life cycle of the scypohozoan jellyfish Aurelia aurita.

BACKGROUND: The moon jellyfish Aurelia aurita is a widespread scyphozoan species that forms large seasonal blooms. Here we provide the first comprehensive view of the entire complex life of the Aurelia Red Sea strain by employing transcriptomic profiling of each stage from planula to mature medusa. RESULTS: A de novo transcriptome was assembled from Illumina RNA-Seq data generated from six stages throughout the Aurelia life cycle. Transcript expression profiling yielded clusters of annotated transcripts with functions related to each specific life-cycle stage. Free-swimming planulae were found highly enriched for functions related to cilia and microtubules, and the drastic morphogenetic process undergone by the planula while establishing the future body of the polyp may be mediated by specifically expressed Wnt ligands. Specific transcripts related to sensory functions were found in the strobila and the ephyra, whereas extracellular matrix functions were enriched in the medusa due to high expression of transcripts such as collagen, fibrillin and laminin, presumably involved in mesoglea development. The CL390-like gene, suggested to act as a strobilation hormone, was also highly expressed in the advanced strobila of the Red Sea species, and in the medusa stage we identified betaine-homocysteine methyltransferase, an enzyme that may play an important part in maintaining equilibrium of the medusa's bell. Finally, we identified the transcription factors participating in the Aurelia life-cycle and found that 70% of these 487 identified transcription factors were expressed in a developmental-stage-specific manner. CONCLUSIONS: This study provides the first scyphozoan transcriptome covering the entire developmental trajectory of the life cycle of Aurelia. It highlights the importance of numerous stage-specific transcription factors in driving morphological and functional changes throughout this complex metamorphosis, and is expected to be a valuable resource to the community.

Lysyl oxidase-like-2 promotes tumour angiogenesis and is a potential therapeutic target in angiogenic tumours.

Lysyl oxidase-like 2 (LOXL2), a secreted enzyme that catalyzes the cross-linking of collagen, plays an essential role in developmental angiogenesis. We found that administration of the LOXL2-neutralizing antibody AB0023 inhibited bFGF-induced angiogenesis in Matrigel plug assays and suppressed recruitment of angiogenesis promoting bone marrow cells. Small hairpin RNA-mediated inhibition of LOXL2 expression or inhibition of LOXL2 using AB0023 reduced the migration and network-forming ability of endothelial cells, suggesting that the inhibition of angiogenesis results from a direct effect on endothelial cells. To examine the effects of AB0023 on tumour angiogenesis, AB0023 was administered to mice bearing tumours derived from SKOV-3 ovarian carcinoma or Lewis lung carcinoma (LLC) cells. AB0023 treatment significantly reduced the microvascular density in these tumours but did not inhibit tumour growth. However, treatment of mice bearing SKOV-3-derived tumours with AB0023 also promoted increased coverage of tumour vessels with pericytes and reduced tumour hypoxia, providing evidence that anti-LOXL2 therapy results in the normalization of tumour blood vessels. In agreement with these data, treatment of mice bearing LLC-derived tumours with AB0023 improved the perfusion of the tumour-associated vessels as determined by ultrasonography. Improved perfusion and normalization of tumour vessels after treatment with anti-angiogenic agents were previously found to improve the delivery of chemotherapeutic agents into tumours and to result in an enhancement of chemotherapeutic efficiency. Indeed, treatment with AB0023 significantly enhanced the anti-tumourigenic effects of taxol. Our results suggest that inhibition of LOXL2 may prove beneficial for the treatment of angiogenic tumours.

Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.

Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -

A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion.

BACKGROUND: The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. METHODS: We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively. RESULTS: The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2) is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells. CONCLUSION: Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on tumor cell invasiveness.