Physiological tolerance of the early life history stages of fresh water prawn (Macrobrachium rosenbergii De Man, 1879) to environmental stress

382-389The present study examines the effect of temperature and salinity on the larval development and survival of Macrobrachium rosenbergii. The larvae showed 100 % mortality at higher temperature (33.5 ± 0.5 °C) in all the salinity conditions (12, 15, and 20 PPT). The survival rate varied between 76-96 % on exposure to lesser temperature conditions. Likewise, the post-embryonic yolk lasted for 4 days at ambient temperature (29 °C); whereas, at 33.5 ± 0.5 °C, it lasted only for 2-3 days. There was an increase in total length of larvae, when exposed to higher temperature and salinity. For the cardiac performance, larval heart beat (fH) significantly increased for higher temperature and salinity conditions (20 PPT; 33.5 °C) and lowered at ambient condition 12 PPT; 29 °C. Larval stroke volume Vs, Cardiac output (Qt) were higher in ambient conditions and lowest in higher temperature and salinity conditions. The larval activity decreased significantly at higher temperature and salinity conditions, compared to ambient conditions

A Novel Cold-Regulated Cold Shock Domain Containing Protein from Scallop Chlamys farreri with Nucleic Acid-Binding Activity

Background: The cold shock domain (CSD) containing proteins (CSDPs) are one group of the evolutionarily conserved nucleic acid-binding proteins widely distributed in bacteria, plants, animals, and involved in various cellular processes, including adaptation to low temperature, cellular growth, nutrient stress and stationary phase. Methodology: The cDNA of a novel CSDP was cloned from Zhikong scallop Chlamys farreri (designated as CfCSP) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full length cDNA of CfCSP was of 1735 bp containing a 927 bp open reading frame which encoded an N-terminal CSD with conserved nucleic acids binding motif and a C-terminal domain with four Arg-Gly-Gly (RGG) repeats. The CSD of CfCSP shared high homology with the CSDs from other CSDPs in vertebrate, invertebrate and bacteria. The mRNA transcripts of CfCSP were mainly detected in the tissue of adductor and also marginally detectable in gill, hepatopancreas, hemocytes, kidney, mantle and gonad of healthy scallop. The relative expression level of CfCSP was up-regulated significantly in adductor and hemocytes at 1 h and 24 h respectively after low temperature treatment (P,0.05). The recombinant CfCSP protein (rCfCSP) could bind ssDNA and in vitro transcribed mRNA, but it could not bind dsDNA. BX04, a cold sensitive Escherichia coli CSP quadruple-deletion mutant, was used to examine the cold adaptation ability of CfCSP. After incubation at 17uC for 120 h, the strain of BX04 containing the vector pINIII showed growth defect and failed to form colonies, while strain containing pINIII-CSPA or pINIII

A C1q domain containing protein from scallop Chlamys farreri serving as pattern recognition receptor with heat-aggregated IgG binding activity.

BACKGROUND: The C1q domain containing (C1qDC) proteins refer to a family of all proteins that contain the globular C1q (gC1q) domain, and participate in a series of immune responses depending on their gC1q domains to bind a variety of self and non-self binding ligands. METHODOLOGY: In the present study, the mRNA expression patterns, localization, and activities of a C1qDC protein from scallop Chlamys farreri (CfC1qDC) were investigated to understand its possible functions in innate immunity. The relative expression levels of CfC1qDC mRNA in hemocytes were all significantly up-regulated after four typical PAMPs (LPS, PGN, β-glucan and polyI:C) stimulation. During the embryonic development of scallop, the mRNA transcripts of CfC1qDC were detected in all the stages, and the expression level was up-regulated from D-hinged larva and reached the highest at eye-spot larva. The endogenous CfC1qDC was dominantly located in the hepatopancreas, gill, kidney and gonad of adult scallop through immunofluorescence. The recombinant protein of CfC1qDC (rCfC1qDC) could not only bind various PAMPs, such as LPS, PGN, β-glucan as well as polyI:C, but also enhance the phagocytic activity of scallop hemocytes towards Escherichia coli. Meanwhile, rCfC1qDC could interact with human heat-aggregated IgG, and this interaction could be inhibited by LPS. CONCLUSIONS: All these results indicated that CfC1qDC in C. farreri not only served as a PRR involved in the PAMPs recognition, but also an opsonin participating in the clearance of invaders in innate immunity. Moreover, the ability of CfC1qDC to interact with immunoglobulins provided a clue to understand the evolution of classical pathway in complement system

The Gln32Lys polymorphism in HSP22 of Zhikong scallop Chlamys farreri is associated with heat tolerance.

BACKGROUND: Heat shock protein 22 is a member of small heat shock proteins with molecular chaperone activity. Though their multiple functions have been well characterized, there is no report about the association between the polymorphisms of HSP22 and heat tolerance. METHODOLOGY: Three single nucleotide polymorphisms were identified in HSP22 from scallop Chlamys farreri (CfHSP22), and the +94 C-A locus was found to be nonsynonymous. Three genotypes at locus +94, A/A, A/C and C/C, were revealed by using Bi-PASA PCR analysis, and their frequencies were 19.5%, 27.6% and 52.9% in the heat resistant stock, while 9.3%, 17.4% and 73.3% in the heat susceptible stock, respectively. The frequency differences of the three genotypes were significant (P<0.05) between the two stocks. After incubating at 30°C for 84 h, the cumulative mortality of scallops with +94 C/C genotype and +94 A/C genotypes was 95% and 90%, respectively, which was significantly higher (P<0.01) than that of scallops with +94 A/A genotype (70%). The molecular chaperone activity of two His-tagged fusion proteins, rCfHSP22Q with +94 C/C genotype and rCfHSP22K with +94 A/A genotype were analyzed by testing the ability of protecting citrate synthase (CS) against thermal inactivation in vitro. After incubated with rCfHSP22Q or rCfHSP22K at 38°C for 1 h, the activity of CS lost 50% and 45%, and then recovered to 89% and 95% of the original activity following 1 h restoration at 22°C, respectively, indicating that the mutation from Gln to Lys at this site might have an impact on molecular chaperone activities of CfHSP22. CONCLUSIONS: These results implied that the polymorphism at locus +94 of CfHSP22 was associated with heat tolerance of scallop, and the +94 A/A genotype could be a potential marker available in future selection of Zhikong scallop with heat tolerance

ELISA analysis of the interaction between rCfC1qDC and human heat-aggregated IgG.

<p>Plates were coated with various PAMPs several concentrations of rCfC1qDC and rTrx, and then incubated with human heat-aggregated IgG. The interaction was detected with goat–anti-human Ig-alkaline phosphatase conjugate at 405 nm. Results are representative of average three such experiments.</p

SDS-PAGE and western-blot analysis of rCfC1qDC.

<p>Lane 1: protein molecular standard (kDa); lane 2: negative control for rCfC1qDC (without induction); lane 3: induced rCfC1qDC; lane 4: purified rCfC1qDC; lane 5: western blot based on the sample of line 3.</p

ELISA analysis of the interaction between rCfC1qDC and the PAMPs.

<p>Plates were coated with various PAMPs, and then incubated with several concentrations of rCfC1qDC and rTrx at 18°C for 3 h. After incubated with rat polyclonal antiserum, the interaction was detected with goat–anti-rat Ig-alkaline phosphatase conjugate at 405 nm. Samples with P/N >2.1 were considered positive. Results are representative of average three such experiments.</p

Temporal expression of the CfC1qDC transcripts in hemocytes after PAMPs challenge.

<p>Vertical bars represent the mean ± S.E. (N = 5).</p