Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and several bacterial challenges in mussels

Cellura, Cinzia; Toubiana, Mylène; Parrinello, Nicolò; Roch, Philippe

doi:10.1016/j.fsi.2006.06.007

Cited by 77 publications

(69 citation statements)

References 40 publications

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“…a totally different technique eliminating eventual bias due to q-PCR technology . We controlled that down regulation of myticin and up regulation of defensin (data not shown) evolved in the present bacterial challenged samples as previously observed (Cellura et al, 2007), validating the general down regulation we reported for Mg-MIF. Although difficult to understand, Mg-MIF does not behave as in other animal models.…”

Section: Coding Sequencessupporting

confidence: 91%

“…Negative control reactions contained water in place of cDNA template and were included in each run to ensure absence of contamination. House keeping gene expression was represented by 28S ribosomal RNA, using the forward primer 5 0 -AAGCGAGAAAAGAAACTAAC-3 0 and the reverse primer 5 0 -TTTACCTCTAAGCGGTTTCAC-3 0 , as previously validated (Cellura et al, 2007;Li et al, 2008) with an annealing temperature of 65°C. Calibration curves were obtained using 10-fold serial dilutions of MIF or 28S rRNA amplicons in 10 lg/ ml sonicated salmon sperm DNA (Sigma).…”

Section: Q-pcr Using the Sybr Green Chemistrymentioning

confidence: 99%

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MIF from mussel: Coding sequence, phylogeny, polymorphism, 3D model and regulation of expression

Parisi

Toubiana

Mangano

et al. 2012

Developmental & Comparative Immunology

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a b s t r a c tThree macrophage migration inhibitory factor (MIF)-related sequences were identified from a Mytilus galloprovincialis EST library. The consensus sequence included a 5 0 -UTR of 32 nucleotides, the complete ORF of 345 nucleotides, and a 3 0 -UTR of 349 nucleotides. As for other MIFs, M. galloprovincialis ORF does not include any signal or C-terminus extensions. The translated sequence of 115 amino acids possesses a molecular mass of 12,681.4, a pI of 6.27 and a stability index of 21.48. Its 3D structure resembles human MIF except for one shorter a-helix. Although evolutionary separated from ticks and vertebrates, Mg-MIF appeared to be closely related to Pinctada fucata and Haliotis, but not to Chlamys farreri and Biomphalaria glabrata. Numerous mutation points were observed within the Mg-MIF ORF, defining 11 amino acid variants within the mussels from Palavas-France and 14 amino acid variants within the mussels from Palermo-Italy. The 2 major variants from Palavas were identical to 2 of the 4 major variants from Palermo. In all the 18 Mg-MIF variants, residues involved in tautomerase and in oxidoreductase activities were conserved. Generally, one mussel expressed 2 Mg-MIF amino acid sequences but with different frequencies of occurrence. Mg-MIF is constitutively expressed principally in hemocytes and in the mantle. In contrast to other animal models, Mg-MIF expression was always down regulated following challenge by bacteria and fungi, confirming previous data obtained with microarray. Down regulation started as soon as 1 h and Mg-MIF expression returned to background 9-48 h after the challenge. Exception was regarding the yeast, Candida albicans, down-regulation between 9 and 72 h, suggesting yeast and bacteria-filamentous fungi trigger different mechanisms of elimination.

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Section: Coding Sequencessupporting

confidence: 91%

Section: Q-pcr Using the Sybr Green Chemistrymentioning

confidence: 99%

MIF from mussel: Coding sequence, phylogeny, polymorphism, 3D model and regulation of expression

Parisi

Toubiana

Mangano

et al. 2012

Developmental & Comparative Immunology

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“…Nonetheless, haemolymph (including plasma and/ or hemocytes) is often chosen as target in stress research using invertebrate organisms as models (e.g., Joy and Gopinathan 1995;Guo et al 2004;Cellura et al 2007;De la Veja et al 2007;Díaz et al 2010;Chen et al 2010;Liu et al 2011). Haemolymph samples were centrifuged for 5 min at 16,000×g and then diluted 1:100 in 0.05 M carbonate-bicarbonate buffer (Sigma-Aldrich, St. Louis, MO).…”

Section: Hsp70 Extraction and Quantificationmentioning

confidence: 99%

Thermal tolerance of the crab Pachygrapsus marmoratus: intraspecific differences at a physiological (CTMax) and molecular level (Hsp70)

Madeira

Narciso

Cabral

et al. 2012

Cell Stress and Chaperones

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Temperature is one of the most important variables influencing organisms, especially in the intertidal zone. This work aimed to test physiological and molecular intraspecific differences in thermal tolerance of the crab Pachygrapsus marmoratus (Fabricius, 1787). The comparisons made focused on sex, size, and habitat (estuary and coast) differences. The physiological parameter was upper thermal limit, tested via the critical thermal maximum (CTMax) and the molecular parameter was total heat shock protein 70 (Hsp70 and Hsp70 plus Hsc70) production, quantified via an enzyme-linked imunosorbent assay. Results showed that CTMax values and Hsp70 production are higher in females probably due to different microhabitat use and potentially due to different hormonal regulation in males and females. Among females, non-reproducing ones showed a higher CTMax value, but no differences were found in Hsp70, even though reproducing females showed higher variability in Hsp70 amounts. As reproduction takes up a lot of energy, its allocation for other activities, including stress responses, is lower. Juveniles also showed higher CTMax and Hsp70 expression because they occur in greater shore heights and ageing leads to alterations in protein synthesis. Comparing estuarine and coastal crabs, no differences were found in CTMax but coastal crabs produce more Hsp70 than estuarine crabs because they occur in drier and hotter areas than estuarine ones, which occur in moister environments. This work shows the importance of addressing intraspecific differences in the stress response at different organizational levels. This study shows that these differences are key factors in stress research, climate research, and environmental monitoring.

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“…In fact, the so-called antifungal activity has been tested only against filamentous fungi: Neurospora crassa for M. edulis [7] and F. oxysporum for M. galloprovincialis [31]. Challenge with Gram-positive M. lysodeikticus resulted in general down regulations of AMP gene expression [5]. Gram-negative challenge effect was more complex with rapid up-regulation (from 1 to 24 h) of defensin by V. splendidus, to late up regulation (from 1 to 3 days) of mytilin by V. anguillarum.…”

Section: Discussionmentioning

confidence: 99%

“…Primers for house keeping gene 28S rRNA, mytilin and myticin mRNA quantifications were from previous report [5] Gene expression levels were quantified by the Livak (2 ÀDDCT ) method using the ratio of equivalent target amount (ETA) for the considered gene on ETA for 28S rRNA. Internal reference of 28S expression was measured in each time point as recommended [10].…”

Section: Quantitative Pcr (Q-pcr)mentioning

confidence: 99%

Gene expression specificity of the mussel antifungal mytimycin (MytM)

Sonthi

Cantet

Toubiana

et al. 2012

Fish & Shellfish Immunology

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a b s t r a c tWe previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR in hemocytes. By contrast, injection of the filamentous fungus, Fusarium oxysporum, induced a sudden and strong increase of expression at 9h p.i. (stimulation index of 25.7 AE 2.1). Optimum stimulating dose was 10 4 spores of F. oxysporum per mussel. In the same samples, AMP mytilin and myticin showed no stimulation. Consequently, we hypothesized the existence of 2 different signal transduction pathways, one activated by bacteria and yeast, the other triggered by filamentous fungi. A second challenge performed with F. oxysporum 24 h after the first challenge induced an increase of MytM gene expression (stimulation index of 3.5 AE 1.7). However, this second increase was significantly lower than the first, suggesting less efficient response rather than significant protection.

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Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and several bacterial challenges in mussels

Cited by 77 publications

References 40 publications

MIF from mussel: Coding sequence, phylogeny, polymorphism, 3D model and regulation of expression

MIF from mussel: Coding sequence, phylogeny, polymorphism, 3D model and regulation of expression

Thermal tolerance of the crab Pachygrapsus marmoratus: intraspecific differences at a physiological (CTMax) and molecular level (Hsp70)

Gene expression specificity of the mussel antifungal mytimycin (MytM)

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