Ahmed Siah

Identification and expression of immune-related genes in hemocytes of soft-shell clams, Mya arenaria, challenged with Vibrio splendidus

Although the mollusc immune system has been studied at the cellular level, the response to pathogens at gene expression level has not been thoroughly investigated. This study aimed to investigate the early molecular response of hemocytes of soft-shell clams, Mya arenaria, to Vibrio splendidus strain LGP32 by identification of transcripts involved in immune defense. The Suppression Subtractive Hybridization (SSH) was used to selectively identify differentially expressed genes in hemocytes exposed to V. splendidus at a ratio 1:1 for 2 h. Both forward and reverse subtracted cDNA were constructed and a total of 16,000 reads were obtained and analyzed. Identity searches in genome databases were performed using BlastX program and transcripts were clustered to cellular functions including structural proteins, immunity, stress proteins, apoptosis, cell process, metabolism and signal transduction. Among the differentially expressed immune associated genes were ficolin, killer cell lectin-like receptor, natural resistance-associated macrophage protein 1 (Nramp-1), mitogen-activated protein kinases (MAPK), ferritin, heat shock proteins 90 (HSP90) and cathepsin and their expressions were quantified using Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) at 1, 2 and 3 h post-Vibrio challenge. These genes showed similar expression patterns, up-regulation at 1 h, followed by a down-regulation at 2 and 3 h. These data corroborates our previous observations of cell rounding, reduced phagocytosis and respiratory burst activity. To our knowledge, this is the first study to demonstrate an effect of V. splendidus on expression of genes related to immune system in soft-shell clams M. arenaria. However, further investigations are needed to unravel the molecular mechanisms of hemocytes subjected to V. splendidus.

Differential gene expression of γ-actin, Toll-like receptor 2 (TLR-2) and interleukin-1 receptor-associated kinase 4 (IRAK-4) in Mya arenaria haemocytes induced by in vivo infections with two Vibrio splendidus strains.

Immune function gene expression in Mya arenaria haemocytes was evaluated following in vivo infection with Vibrio splendidus LGP32-GFP and 7SHRW. Elongation factor 1alpha (EF-1alpha) with 2 (EF-2), after challenge with LGP32-GFP, and EF-1alpha with the ribosomal protein S-18, after challenge with 7SHRW, were found to be the most stable housekeeping genes. Using these internal controls and comparing the regulation induced by both strains, up-regulation of gamma-actin, down-regulation of TLR-2 and up-regulation of IRAK-4 was significantly higher after challenge with LGP32-GFP (p<0.001, p=0.001 and p<0.05, respectively). These results suggest specific responses at a molecular level modulated by the bacterial strains. LGP32-GFP induced marked responses which coincide with a similar trend previously found on phenotypic responses under our experimental model.

Selection and evaluation of housekeeping genes for haemocytes of soft-shell clams (Mya arenaria) challenged with Vibrio splendidus.

Gene expression studies have opened a tremendous field of investigation in biological research over the last decades. Expression of genes is most frequently quantified by real time PCR (RT-qPCR), as this method has proven to be highly sensitive. One of the critical steps, however, in comparing transcription profiles is the availability of selected housekeeping genes. Expression of these genes should be steadily stable across the conditions under study so that they provide a baseline for gene expression comparison. Such a baseline is best established using a set of few housekeeping genes. Usually, those genes are involved in maintaining homeostasis and cell viability. In our study, nine candidate genes were used, including some commonly used housekeeping genes, such as ribosomal RNA (18S, S-15, S-18 and L-37), beta actin, ubiquitin, receptor activated C kinase (RACK) and elongation factor 1 and 2, in order to determine the most stable housekeeping genes, after haemocytes of Mya arenaria were exposed to Vibrio splendidus for 2 h. Our results showed that EF-1, S-18 and ubiquitin appear to be the most stable genes for this experimental condition. On the other hand, both 18S and beta actin, the most widely used housekeeping genes, turned out to be the least stable. This demonstrates the absolute need for preliminary assessment of housekeeping genes in gene expression studies.

Selecting a set of housekeeping genes for quantitative real-time PCR in normal and tetraploid haemocytes of soft-shell clams, Mya arenaria

The transcripts involved in the molecular mechanisms of haemic neoplasia in relation to the haemocyte ploidy status of the soft-shell clam, Mya arenaria, have yet to be identified. For this purpose, real-time quantitative RT-PCR constitutes a sensitive and efficient technique, which can help determine the gene expression involved in haemocyte tetraploid status in clams affected by haemic neoplasia. One of the critical steps in comparing transcription profiles is the stability of selected housekeeping genes, as well as an accurate normalization. In this study, we selected five reference genes, S18, L37, EF1, EF2 and actin, generally used as single control genes. Their expression was analyzed by real-time quantitative RT-PCR at different levels of haemocyte ploidy status in order to select the most stable genes. Using the geNorm software, our results showed that L37, EF1 and S18 represent the most stable gene expressions related to various ploidy status ranging from 0 to 78% of tetraploid haemocytes in clams sampled in North River (Prince Edward Island, Canada). However, actin gene expression appeared to be highly regulated. Hence, using it as a housekeeping gene in tetraploid haemocytes can result in inaccurate data. To compare gene expression levels related to haemocyte ploidy status in Mya arenaria, using L37, EF1 and S18 as housekeeping genes for accurate normalization is therefore recommended.

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: changes in cell structure, numbers and adherence.

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p<0.01) than with 7SHRW. The cell adherence was markedly diminished (p<0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p<0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.

Changes induced by two strains of Vibrio splendidus in haemocyte subpopulations of Mya arenaria, detected by flow cytometry with LysoTracker

Flow-cytometric characterisation of bivalve haemocytes is usually performed by light-scatter profiles based on size and complexity of the cells. Additional means of characterisation such as specific fluorescent dyes are not commonly used to discriminate cell subpopulations in challenged and unchallenged haemocytes. In the present study, we characterise the changes in haemocyte subpopulations of soft-shell clam Mya arenaria induced by in vivo challenge with 2 strains of Vibrio splendidus by using a fluorescent probe. Responses were measured 24 h after infection with either a local wild strain (7SHRW) or a modification (LGP32-GFP) of a strain associated with oyster mortalities in France (LGP32). Changes in haemocyte subpopulations were analysed using flow cytometry based on 2-parameter scatter profiles and lysosomal content reflected by LysoTracker staining. Forward and side-scatter profiles revealed 2 haemocyte subpopulations: hyalinocytes and granulocytes. Granulocytes exhibited significantly higher levels of lysosomal staining (p < 0.01). Following infection with LGP32-GFP, both subpopulations merged into a single continuous group and their lysosomal content significantly decreased (p < 0.05). Independent modifications after infection were observed in the proportions of subpopulations established by their lysosomal content. While the subpopulation of hyalinocytes had lower levels of lysosomal content after infection, especially with LGP32-GFP (p < 0.001), the subpopulation of granulocytes had similar levels of lysosomes after infection with 7SHRW and significantly decreased levels after infection with LGP32-GFP (p = 0.001). Our data suggest specific modulation of bivalve responses against pathogenic bacteria that would include degranulation.

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r(2)=0.68, p<0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.

Functional and molecular responses in Mytilus edulis hemocytes exposed to bacteria, Vibrio splendidus

This study aims at examining the morphological, functional and molecular responses of Mytilus edulis hemocytes exposed to different strains of Gram-negative bacteria Vibrio splendidus (a virulent strain V. splendidus LGP32, V. splendidus LGP32 Δvsm without metalloprotease and an environmental type strain V. splendidus 7SHRW) at a 1:3 ratio for 2, 4, and 6 h. Our data showed that hemocytes could have a discriminative capacity towards microorganisms. Both V. splendidus LGP32 strains had an effect on hemocyte adhesion, phagocytosis abilities and oxidative burst, whereas the environmental strain 7SHRW induced weak and delayed hemocyte responses. At a molecular level, differential levels of candidate transcripts were measured in M. edulis hemocytes exposed to V. splendidus LGP32-GFP and 7SHRW. Mainly, a down-regulation of defensin was recorded in hemocytes exposed to V. splendidus LGP32. A significant up-regulation of lysozyme and proteasome 26S was observed at 2 h followed by a down-regulation at 4 and 6 h of exposure to the LGP32 strain. Similarly, SOD and GPx genes were up-regulated 2 h post-exposure to LGP32 strain and their expressions decreased after 4 and 6 h post-exposure. Further analysis is however needed in a near future to relate the transcript level variations with the physiological process.

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

MORPHOLOGICAL AND MOLECULAR EFFECTS OF VIBRIO SPLENDIDUS ON HEMOCYTES OF SOFTSHELL CLAMS, MYA ARENARIA

ABSTRACT Hemocytes constitute the cellular part of the mollusc immune system and are involved in phagocytosis; the production of toxic oxygen radicals, antimicrobial peptides, opsonizing molecules, and lysozymes; digestion, excretion, and nutrient transport. In this study, we investigated the phenotypic response, phagocytosis, and respiratory burst activity in hemocytes of Mya arenaria exposed to the bacterium Vibrio splendidus. Exposure to V. splendidus led to a loss of pseudopodia and rounding of hemocytes. The phagocytic ability of hemocytes was significantly reduced in challenged hemocytes, as was the respiratory burst activity of hemocytes. The expression of actin and elongation factor 2 genes was measured to investigate a possible relation between phenotypic response of hemocytes exposed to V. splendidus and genes associated with cytoskeleton. Both actin and elongation factor 2 genes were upregulated in challenged hemocytes. Additional studies are underway to identify other genes of hemocytes whose expression is affected by exposure to V. splendidus.