Dietary Lipid Requirement Of Broodstock Red Claw Crayfish Cherax Quadricarinatus And The Function Of Fatty Acid Binding Protein

Since its introduction in China in 1992, demand for the red claw crayfish (Cherax quadricarinatus), a popular commercial aquaculture crustacean species, has increased dramatically due to its large size and positive marketing potential. In China, red claws are often fed commercial prawn feeds which despite their high price fail to meet the nutritional demands of the red claw. Feed is often one of highest costs in an aquaculture enterprise, and though diets that promote red claw growth is of growing interest to producers, little attention has been placed on developing adequate diets for maturing broodstock. Thus, nutritionally tailored and economical feed formulations are needed for red claws in aquaculture. In current study, the effects of four dietary lipid sources on growth and gonad maturation of pre-adult red claws were evaluated first. The effects of different cholesterol levels in feed were evaluated by the same way. To investigate the modulation function of different fatty acids, the fatty acid binding protein Cq-FABP was cloned and expressed. Some bio-photonics approaches were employed to study the ligands binding properties of Cq-FABP.1. Effect of different dietary lipid sources on growth and biochemical composition of pre-adult female Cherax quadricarinatusThe effects of four dietary lipid sources (fish oil, peanut oil, soybean oil, pork lard) on growth and biochemical composition of pre-adult female red claw crayfish, Cherax quadricarinatus, were evaluated. Performance was evaluated by weight gain, specific growth rate (SGR), survival, feed conversion ratio (FCR), gonadosomatic index (GSI) and fatty acid composition. The proportion ofsaturated fatty acids (SFA), mono-unsaturated fatty acids (MUFA), and poly-unsaturated fatty acids (PUFA) in hepatopancreas varied as a result of lipid source, while differences in muscle were limited to MUFA levels. The optimal growth observed in red claws receiving the soybean oil diet. Conversely, diets high in EPA (20:5n3) and DHA (22:6n3) produced suboptimal results, suggesting that the importance of maturation diets with high EPA and DHA content may be exaggerated. As the dietary lipid requirements of growing and reproductively active red claw crayfish were satisfied by a plant oil that contained high levels of 18-carbon unsaturated fatty acids, soybean oil may be an economic alternative to the expensive lipid-fortified feed currently utilized by the aquaculture industry.2. Effect of different dietary lipid sources on digestive enzyme activities and Vg expression of pre-adult female Cherax quadricarinatusThe effects of four dietary lipid sources (fish oil, peanut oil, soybean oil, pork lard) on digestive enzyme activities and Vg expresson of pre-adult female red claw crayfish, Cherax quadricarinatus, were evaluated. The activities of digestive enzymes were influenced by different dietary lipid sources, differed significantly. The soybean oil group has a higher activities than those from the other treatments. The low activities in red claws receiving the diet 4 possibly reflecting a lack of essential fatty acids in pork lard. Since GSI increases slowly at the onset of ovarian maturation, differences in dietary lipid sources did not alter GSI in present study, while the change in the hepatopancreatic Vg expression is significant. The Vg expression, with peak expression observed in red claws receiving the soybean oil diet, indicated that the fatty acids in soybean oil may have the function on stimulating the gonad maturation.3. Effect of different dietary cholesterol levels on growth and reproduction of female pre-adult Cherax quadricarinatusThe effect of dietary cholesterol level on growth and reproduction performance of female pre-adult red claw crayfish Cherax quadricarinatus was evaluated over a 60-day culture period. Five experimental diets, supplemented with 0%,0.25%,0.50%, 0.75% and 1.00% cholesterol were evaluated. The growth performance, digestive enzyme activities and Vg expression in hepatopancreas were measured. A quadratic equation was used for regression analysis which was used to determine the digestive enzyme activities response to the dietary cholesterol level. The results showed that the female red claw crayfish fed with the dietary cholesterol ranged from 0.25-0.50% had better weight gain, SGR, survival rate, and FCR, and higher Lipase and Trypsin activities than those from the other treatments. The regression results from the growth performance and the digestive enzyme activities showed that the optimum cholesterol content was 0.55%. Female red claw crayfish fed with the diet without cholesterol supplementation had the lowest growth, digestive enzyme activities and the Vg expression. Though the group 5 (1% cholesterol) had the highest Vg expression, consider the overall performance, the recommended dietary cholesterol level for optimum growth and gonad maturation for female red claw crayfish under these environmental conditions was 0.55%. During rapid gonadal development phase in female C. quadricarinatus, a 0.75% cholesterol diet was recommended.4. Molecular cloning and sequence analysis of the fatty acid binding protein (Cq-FABP) gene in female red claw crayfish Cherax quadricarinatusA cDNA encoding red claw crayfish fatty acid binding protein (Cq-FABP) gene was cloned by using rapid amplification of cDNA ends (RACE). The full length cDNA was 772 bp, containing a 82 bp 5’-UTR region, a 291bp 3’-UTR region and a 399bp ORF region which encoded a 132 aa polypeptide. The secondary and tertiary structure assay showed that the Cq-FABP has 10β-strands and two a-helices, which is delimiting a cavity where the hydrophobic ligands are bound just as other FABPs. The deduced amino acids sequence of Cq-FABP sharing 90.9% homology with P.leniusculus and 56.8%,56.8%,53.8%,55.3%,53.8% with E.sinensis, P.clarkii, M.ensis, L.vannamei, P.monodon respectively. The homology with FABPs of human were lower, sharing 37.0%,35.4%,35.4%,38.6%,37.0%,40.9%,31.5%,38.6% homology with A-FABP, S-FABP, E-FABP, H-FABP, B-FABP, L-FABP, M-FABP, respectively. The high homology of Cq-FABP with B-FABP implied that they may have a close function on modulation of gene transcription. A NJ poylogenetic tree was also constructed based on reported FABPs amino acid sequences, results showed that the tree topology is agreement with traditional taxonomic relationships. The Decapoda species were in the same cluster, but the Cq-FABP together with the FABP from P.leniusculus were clustered in a different group with Decapoda species.5. Expression pattern of the fatty acid binding protein (Cq-FABP) gene in female red claw crayfish Cherax quadricarinatusA real-time qRT-PCR was employed to investigate the distribution of Cq-FABP in different tissues as well as to assess expression in hepatopancreas fed diets with different lipid sources. The beta-actin was used to normalize the Cq-FABP expression. Results showed that Cq-FABP was widely distributed, with the highest expression level in hepatopancreas, small amount of expression in intestine, detectable expression level in ovary, antennal gland, hemolymph, gills, stomach and testis, while expression was almost undetectable in muscle. The distribution manner indicated the possible function of Cq-FABP mainly involved in the intake and utilization of fatty acids. The Cq-FABP expression pattern in hepatopancreas with different lipid source diets treatment showed the influence of fatty acids on the Cq-FABP expression levels, the expression level seems relate to the amount of the long chain fatty acids in the diets, except the soybean oil group. Considering the good growth and high Vg expression level, the different expression pattern of Cq-FABP in soybean oil group implied the Cq-FABP may involved in gene modulation. 6. Expression, purification and fatty acids binding properties of the recombinant Cq-FABPThe Fragment contained the complete coding sequence of the Cq-FABP with NdeⅠand BamHⅠwas obtained by PCR, and an expression vector pET15b-His6-Cq-FABP has been constructed. The 15 kDa Cq-FABP protein was successfully expressed by the constructed Cq-FABP expression strain after the IPTG induction.Interaction of various fatty acids with recombinant Cq-FABP was studied by different fluorescence procedures. In order to characterize the binding of several non fluorescent fatty acids (the dominant fatty acid in different lipid source diets) to Cq-FABP, we first indirectly determined their apparent affinities by an equilibrium displacement approach using ANS by using the steady-state fluorescence emission spectra. After correction by both the inner filter effect and the "non specific" binding of ANS, the Kd of palmitic acid (PA) was calculated from linear regression plotted by a series of competitive titrations. Next, a fluorescence resonance energy transfer (FRET) assay was conducted to address the binding of palmitic acid (PA) to Cq-FABP. TheΔ%FRET values were measured both by the steady-state fluorescence measurements and time-resolved fluorescence measurements. By plotting both the fluorescence intensity parameter and the FRET efficiency parameter, the calculated Kd,PA values were compatible with the one determined by monitoring the ANS fluorescence. After comparing the R-square of the two procedures, the Kd values of other fatty acids were measured by the first one. Results showed that the Kd values for Palmitic acid (PA), Oleic acid (OA), linoleic acid (LA), Eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-Docosahexaenoic acid (DHA) were 1.1,2.4,3.1,2.9 and 5.8 respectively. Then the BODIPY-labeled fatty acids BODIPY C16 which does not have the "non-specific" with Cq-FABP was employed to measure the Kd values by steady-state fluorescence anisotropy based assay. The the Kd values for PA, OA, LA, EPA and DHA were 1.8,2.7,3.8,3.7 and 7.2 respectively, which were compatible with the value obtained by the previous procedure. Based on the Kd values of fatty acids, the Cq-FABP expression profile in the treatments with different lipid sources indicated that the high Vg expression may due to the LA, which may have the function of modulating the gene expression by the manner of regulating the Cq-FABP.