Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata

Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO₃) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.     

First molluscan TNF-alpha homologue of the TNF superfamily in disk abalone: molecular characterization and expression analysis

Tumor necrosis factor alpha (TNF-alpha) is considered as a multifunctional immune modulator that plays an important role in the innate and adaptive immune systems in vertebrates. Here, we described the characterization and expression analysis of the first TNF-alpha homologue in mollusk abalone, named as AbTNF-alpha. It has 930-bp full length with a 717-bp open reading frame (ORF), encoding 239 amino acids. The AbTNF-alpha amino acid sequence shows the characteristic TNF family signature, N-terminal transmembrane domain consisting of a hydrophobic amino acid cluster and cell attachment sequence at (155)RGD(157). Phylogenic analysis results showed that AbTNF-alpha is more related to the invertebrate Ciona savignyi TNF superfamily ligand member (CsTL). Quantitative real-time PCR expression results showed that AbTNF-alpha was constitutively expressed in both immune and non-immune tissues in a tissue specific manner. The highest constitutive expression was in the gill tissue with a 1.5-fold compared to hemocytes expression. The AbTNF-alpha mRNA expression in gill tissue was monitored in vivo stimulated by a mixture of pathogenic bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Lysteria monocytogenes), viral haemorrhagic septicaemia virus (VHSV) and lipopolysaccharide (LPS). The AbTNF-alpha expression was significantly (p<0.05) induced by bacteria, VHSV and LPS compared to the control animals. Moreover, the highest level expressions of each induction were at 24 h (5.2-fold), 48 h (2.8-fold), and 48 h (3.3-fold) by bacteria mixture, VHSV and LPS, respectively. These results indicate that AbTNF-alpha could respond to pathogenic infection or stimulation and may play an important role in the abalone immune system.     

Transcriptome analysis in the midgut of the earthworm (Eisenia andrei) using expressed sequence tags

In order to gain insight into the expression profiles of the earthworm midgut, we analyzed 1106 expressed sequence tags (ESTs) derived from the earthworm midgut cDNA library. Among the 1106 ESTs analyzed, 557 (50.4%) ESTs showed significant similarity to known genes and represented 229 unique genes of which 166 ESTs were singletons and 63 ESTs manifest as two or more ESTs. While 552 ESTs (49.9%) were sequenced only once, 230 ESTs (20.8%) appeared two to five times and 324 ESTs (29.3%) were sequenced more than five times. Considering this redundancy of expression, it is likely that the gene expression profile of the earthworm midgut would be polarized. The expression of globin-related proteins, including ferritin and linker chain, and fibrinolytic enzymes appeared to account for 10.1% and 4.7% of the total ESTs analyzed in this study, respectively. This suggests that the prime functions of the midgut in the earthworm would be associated with protein hydrolysis as well as globin formation. Among the recognized protein-coding genes, the gene category involved in protein synthesis appeared to be the largest one accounting for 15.6% of the expression in the midgut, followed by gene categories associated with energy (11.2%), homeostasis (10.8%), metabolism (3.6%), cytoskeleton (2.5%), and protein fate (1.4%). With regard to functional aspects, the most abundantly expressed genes were associated with respiratory pigment (10.1%), cellular respiration (8.6%), and fibrin hydrolysis (4.7%). In addition, we were able to identify novel ESTs in the earthworm, which were related to the innate immune system, including destabilase, a possible antagonist of transglutaminase.