Early Development Of Trypsins And Amylases Of Grass Carp Ctenopharyngodon Idellus, Yellowcheck Carp Elopichthys Bambusa And Topmouth Culter Culter Alburnus

The development of digestive function of larval and juvenile fishes has been becoming a key research issue in the digestive physiology of fish. A thorough understanding of the temporal process of structure and function of larval and juvenile digestive systems has important value for investigating the digestive characteristics, nutritional needs of larva and juveniles, and therefore enhancing their survival and growth. More recently, most researches on the digestive function of larvae and juvenile are focused on marine species, and fewer studies have examined the temporal expression patterns of digestive enzymes in freshwater fishes. Cyprinidae accounts for more than half of freshwater fishes in China and constitutes the major group of China’s cultured freshwater fish. In inland aquaculture of China, grass carp Ctenopharyngodon idellus, yellowcheck carp Elopichthys bambusa and topmouth culter Culter alburnus are economically important freshwater species of the family Cyprinidae, with entirely different nutritional habits naturally. Using the methods of histology, examination of enzymatic activities, molecular cloning and quantitative real-time PCR, the comparative study among the above species in early developmental stages has been conducted in the present study to examine the histological development of digestive tracts, the mRNA expression patterns, as well as enzymatic activities of trypsin and a-amylase from the perspectives of feeding habits and phylogeny. Based on the results, the common mode and the differences of feeding habits in the development of digestive function are preliminarily discussed between typically herbivorous and carnivorous cyprinidae fish.The histological results of the three species in different days post hatchting (dph) showed following results:(1) The yellowcheck carp has the maximum relative bulk of yolk sac in newly hatched larva among the three species; (2) The intestinal tubes and oral fissures of three species were differentiated apparently at 2 dph and 3 dph, and intestinal cavities of grass carp, yellowcheck carp and topmouth culter were appeaed at 3 dph,4 dph and 2 dph, respectively; (3) The food pulps were observed inside the intestines of grass carp, yellowcheck carp and topmouth culter in 4 dph,7-9 dph and 4 dph, respectively, which indicated that these larval species had initiated exogenous feeding at different time post hatching; (4) Mucosal folds of intestines in grass carp, yellowcheck carp and topmouth culter were initially differentiated at 5 dph,6 dph and 6 dph, respectively, and subsequently the number and height of mucosal folds has been increased at different degrees with the development of larvae and juveniles, which enables the fishes to increase the total mucosal surface aeras with the development; (5) The intestinal convolution of grass carp and yellowcheck carp was initially observed from the transverse slices at 14 dph and 30 dph respectively, but no intestinal convolution was appeared during the experiment of 30 dph in topmouth culter; (6) The grass carp, yellowcheck carp and topmouth culter were observed to have intestinal mucous cells at 17 dph,30 dph and 20 dph, respectively, and subsequently the numbers of mucous cells increased gradually with development.The full-length cDNA clones encoding trypsinogens for the three species were isolated using RACE PCR and contain 869 bp (accession no. FJ416598),856 bp (not submitted) and 857 bp (accession no. FJ416597), respectively. The open reading frames (ORFs) were 729 bp,741 bp and 744 bp long, and the deduced amino acid sequences were 242 aa,246 aa and 247 aa long, all containing the highly conserved residues essential for serine protease catalytic and conformational maintenance, which confirmed the nucleotide sequences of the trypsinogens being correct. The results from isoelectric and phylogenetic analyses suggest that grass carp trypsinogen is grouped with teleost trypsinogen groupⅠ, while the trypsinogens of yellowcheck carp and topmouth culter are both grouped with groupⅡ.The development of trypsinogen gene expression and tryptic activities of grass carp and topmouth culter were examined by quantitative real-time PCR and specific activity detection of trypsin, the results showed that the expression pattern of trypsinogen mRNA was similar between these two species, appearing at 2 dph and reaching peaks at 11 and 23 dph. The trypsin-specific activities in both species were detected at 2 dph when the larvae did not initiated the first exogenous feeding, then reached the major peaks at 8 dph, however, the minor peaks were observed at 20 dph in the grass carp and 17 dph in the topmouth culter. The trypsin-specific activity was significantly higher in the grass carp than in the topmouth culter, which may be attributed to the nature of their different nutritional habits and this nature probablly behaved during the early developent of lavra.Meanwhile, the full-length of grass carp a-amylse gene cDNA and the partial sequence of yellowcheck carpα-amylse gene cDNA were cloned by RACE PCR. The full-length of grass carpα-amylse gene cDNA was 1616 bp and contained 1539 bp of ORF which encoded 512 aa of amino acid sequence ofα-amylse precursor with 15 aa of signal peptited. The partial sequence of yellowcheck carpα-amylse gene cDNA are 970 bp and contained 927 bp of ORF which encoded 308 aa of amino acid sequence ofα-amylse fragment. This fragment contained theβ4-α4-β5-α5-β6-α6-β7-α7-β8-α8 part of the (β/α)8 barrel which is the central catalytic domain of animalα-amylse. The active site residues(Asp, Glu and Asp) of the two deduced a-amylse sequences were located in the parts ofβ4,β5 andβ7, respectively, which is consistent with other vertebrates’α-amylses. In addition, other highly conserved residues were contained in the twoα-amylse sequences, such as calcium binding residues and chloride binding sites and cysteine residus which form disulfide bonds. These characteristic residues are essential forα-amylse catalytic and conformational maintenance, and conseqently confirmed the nucleotide sequences of the two a-amylses being correct. The results from multiple alignment and isoelectric analyse suggest that a-amylse of grass carp is similar to that of zebra fish Danio rerio in the amino acid sequence composition, and predict isoelectric point and charge at pH 7.0. The phylogenetic analyse suggests that theα-amylse amino acid sequences of grass carp, yellowcheck carp zebra fish and chinese sucker, Myxocyprinus asiaticus, which all belong to cypriniformes, constitute a small group in the phylogenetic tree.The gene expression and specific activities ofα-amylses during the early development in grass carp and yellowcheck carp, which both belong to leuciscina, were investigated. The results showed that theα-amylse expression in both species were detected at 2 dph, but the expression level of yellowcheck was very low. With the development, theα-amylse expression in both species reached the peaks at 7 dph and then decreased. During 11-30 dph, theα-amylse expression of grass carp increased gradually and then maintained a sustainably high level, but that in yellowcheck carp continuously decreased and declined to a very low level at 30 dph. Theα-amylse specific activity of grass carp was detected at 3 dph and reached the peak at 7 dph, and then decreased rapidly, however, the enzymatic activity gradually increased again during 9-30 dph. As for yellowcheck carp, a-amylse specific activity did not detected until 5 dph, but the activity level at this time was so low and approximately corresponded to that of grass carp at 3 dph. Similarly, the a-amylse specific activity of yellowcheck carp increased remarkably at 7 dph, but decreased again during 7-9 dph, then it increased slowly and reached the peak at 16 dph. After 16 dph, the a-amylse specific activity of yellowcheck carp decreased continuously until the end of the experiment. These results suggested that the a-amylse mRNA and its enzyme protein were expressed highly and sustainably around the period of juveniles of grass carp, but in the same phase the expression in yellowcheck carp decreased continuously. The different expression patterns between the two species maybe related closely with their different orientation of feeding habits.