Patty McKenna

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.

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.

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.

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.

Functional and molecular responses of the blue mussel Mytilus edulis' hemocytes exposed to cadmium - An in vitro model and transcriptomic approach

Abstract The bivalve mollusk, Mytilus edulis, is used as a sentinel species in several monitoring programs due to its ability to bio‐accumulate contaminants. Its immune system consists of hemocytes and humoral components, which constitute the main part of the hemolymph. The present study is aimed at understanding the effects of Cd on the differentially expressed genes involved in the phagocytosis of M. edulis’ hemocytes. Our approach focuses on an in vitro model by exposing hemocytes to different concentrations of Cd ranging from 10−9 M to 10−3 M. Phagocytosis and cell viability as functional markers were measured using flow cytometry. The molecular mechanisms regulated by Cd were investigated using RNA‐seq and DGE analysis. Results showed that viability and phagocytosis of hemocytes exposed to 10−3 M of Cd were significantly decreased after 21 h of exposure. RNA sequencing data showed that 1112 transcripts (out of 352,976 contigs) were differentially regulated by the highest concentration of Cd. Among these identified transcripts, 1028 and 84 were up and down‐regulated respectively. The induction of super oxide dismutase (SOD), glutathion‐s‐transferase (GST), cytochrome P450 2C8 (CYP2C8), multidrug resistance protein (MRP1) and heat shock protein 70 (HSP70) suggests that Cd can regulate key molecular mechanisms. In addition, several toll‐like receptors (TLR) as well as genes involved in phagocytosis (actin and CDC42) and apoptosis (caspase 8 and XIAP/IAP) were induced by Cd. Thus, our model highlights the effect of Cd on the phagocytic function of M. edulis’ hemocytes along with the regulation of gene expression involved in innate immunity, detoxification and apoptosis. Further investigations need to be pursued to unravel the effects of Cd on the molecular mechanisms identified in this study. HighlightsTranscriptomic analysis of hemocytes exposed to cadmium.In vitro model of Mytilus edulis hemocytes exposed to cadmium.Molecular mechanisms of hemocytes exposed to cadmium.

Single Plex Branched DNA as an Alternative Assay to Quantify P53-Like mRNA Levels in Softshell Clam Mya arenaria Hemocytes

ABSTRACT Previous studies have shown that hemic neoplasia in softshell clams is related to the level of P53 -like mRNA in hemocytes. Traditionally, the p53-like mRNA level has been quantified using quantitative real-time RT-PCR (Q RT-PCR). However, this technique requires several steps that are sources of contamination and may result in a low accuracy of the analysis. The novel aspect of this study is that the p53-like mRNA level was quantified directly from a lysate of hemocytes without any RNA extraction or reverse transcription steps. This assay is based on branched DNA (bDNA) signal amplification technology and enables quantification of p53-like mRNA levels in as few as 2,500 hemocytes, with a coefficient of variation close to 6% (range, 2–12%). A significant correlation (R2= 0.99, P ≤0.01, n= 5) was found between the p53-like mRNA quantified directly from hemocytes without RNA extraction and from 1 µg total RNA extracted from hemocytes using the classic TRIzol protocol. To compare p53-like mRNA levels in hemocytes from diseased clams collected in North River (Prince Edward Island, Canada) and from healthy clams found in Havre aux Maisons at Magdalene Island (Quebec, Canada), the quantification of relative p53-like mRNA levels was performed using our single plex assay. Data showed a significantly (P ≤0.01, n= 5) high expression of p53-like in the hemocytes of clams collected from North River. Therefore, although Q RT-PCR remains the most widely used technique for the quantification of gene expression level, we believe that single plex using bDNA technology could represent a new generation of mRNA quantification tool, enabling a more efficient mollusc health management.