| Intermuscular bones(IBs)in fish,commonly known as intramuscular spines,are located in the muscle diaphragms on both sides of the vertebrae of the fish body.They are derived from continuous homologous ossification of mesenchymal cells in the connective tissue,and only exist in lower bony fish.They are divided into medullary arch small bones,vein arch small bones,and vertebral body small bones.Most freshwater fish in the world,especially carp fish,generally have numerous and complex intermuscular bones,which hinder consumption,export,deep processing,and seriously affect food and economic value.Acrossocheilus belongs to Cyprinidae.Although it has a large number of intramuscular bones,it is very soft and doesn’t affect consumption and deep processing.Its meat is tender and delicious,and there is a large market demand.However,there is relatively little research on the occurrence and molecular mechanism of the intramuscular bones in Acrossocheilus at home and abroad.In order to study the molecular mechanism of the development of intermuscular bones in Acrossocheilus,this study first conducted morphological identification on the experimental raised Acrossocheilus,and identified the research object as Acrosophilus wenchowsis.During the experiment,artificial breeding was carried out to obtain samples of intermuscular bones at various stages to explore the developmental characteristics of larvae and juveniles.This study observed,statistically analyzed,and analyzed the occurrence period,morphology,and morphology of intermuscular bones in Acrosophilus wenchowsis’s larvae and juveniles,as well as the morphology,number,and distribution of intermuscular bones in adult fish;Based on the three key periods of the development of intermuscular bones,the intermuscular bones and its surrounding muscle tissues were taken for transcriptome analysis to explore the genetic information related to the development of intermuscular bones;Combined with transcriptome sequencing results and KEGG signal pathway analysis,nine genes(bmp2,msx-c,scx,osterix,runx2,bgp,alpl,ost,fgf23)related to and significantly expressed in intermuscular bones were screened out,and their expression levels in three groups of Acrosophilus wenchowsis were analyzed to infer the molecular mechanism of intermuscular bone formation.The main research findings are as follows:1.The traditional morphological measurement methods were used to describe and analyze the morphological characteristics,20 quantifiable traits,and 9 countable traits of Acrossocheilus in the breeding base.The Acrossocheilus was identified as Acrossocheilus wenchowensis.The distinguishing features were:6 vertical lateral lines of melanin stripes on the body side,fixed in position and symmetrical to the left and right;There are differences between the sexes,with female fish having vertical stripes extending downwards beyond the lateral line,while male fish having vertical stripes above the lateral line and a melanin stripe parallel to the lateral line;The flaps on both sides of the lower lip are in contact and cover the mandible,with no keratin at the front edge.The development period of Acrossocheilus wenchowensis’s larvae and juveniles is about 26 days.The early larvae lasted for 7 days,feeding from the yolk sac into the rotifer opening bait;The late stage larvae lasted for 11 days,with 8 vertical melanin bands formed on the body side,and the development of each fin was basically completed;The juvenile stage lasts for 8 days,from the appearance of scales to the full body being covered with scales,with all organs fully developed,and then enters the juvenile stage.2.By staining the overall bones of Acrossocheilus wenchowensis larvae and juveniles,as well as observing and analyzing the morphological anatomy of adult fish,the appearance period,morphology,distribution,and length of intermuscular bones in larvae and juveniles were observed and analyzed.The results showed that:on the 21st day after hatching,the small bones of the medullary arches first appeared in the tail,and then differentiated from the tail to the head in turn.The number of intermuscular bones was 73-97,among which the number of intermuscular bones in the supra-axillary muscle of the trunk was the largest(32-38),the number of intermuscular bones in the infra-axillary muscle of the trunk was the smallest(6-11),the number of intermuscular bones in the supra-axillary and infra-axillary muscles of the tail was similar(16-28),and the length of intermuscular bones in the supra-axillary muscle of the trunk was larger than that in the supra-axillary muscle of the tail.The length of the intermuscular bones in the trunk supra-axillary and caudal subaxillary muscles was greater than that of the intermuscular bones in the trunk subaxillary muscles and significantly greater than that of the intermuscular bones in the trunk subaxillary muscles(p<0.05),there was a significant correlation(p<0.05)between body length and the length of the intermuscular bones in the trunk subaxillary muscles(R2=0.9948),and a significant correlation(p<0.05)between body weight and the length of the intermuscular bones in the trunk supra-axillary muscles(R2=0.9983).There are 6 kinds of intermuscular bones,including "I" "卜" "Y" "(",one-end-multifork,two-end-multifork types,which from simplicity to complexity.3.Based on the three stages of the development of Acrossocheilus wenchowensis’s intermuscular bone(NIB:non long intermuscular bone,LIB:rigid long intermuscular bone,CIB:complete ossification and sclerosis of IBs),transcriptome analysis was carried out on samples of its intermuscular bone and surrounding muscle tissue.The results showed that 173563 unigenes were obtained,of which 33916 were annotated to GO classification,12304 were annotated to 233 KEGG signal pathways.In addition,10166,36554,and 16277 differentially expressed genes were found in the three groups of NIB compared with LIB,NIB compared with CIB,and LIB compared with CIB,respectively,and 211 genes were differentially expressed in all three groups.Further expression pattern clustering analysis and KEGG pathway analysis were performed on it,and 749 genes were annotated into the PI3K-Akt signaling pathway,while 204 genes were annotated into TGF-β 705 genes were annotated to MAPK signaling pathway,308 genes were annotated to Wnt signaling pathway,and some genes were annotated to osteoclast differentiation,metabolism and other signaling pathways.Based on the transcriptome sequencing results and KEGG signal pathway analysis,nine genes(bmp2,msx-c,scx,osterix,runx2,bgp,alpl,ost,fgf23)related to and significantly expressed in intermuscular bones were screened out,and their expression levels in three groups of Acrossocheilus wenchowensis were analyzed.Before and after the growth of intermuscular bones,the bmp2 and msx-c genes begin to express,activating and activating the scx and runx2 genes to regulate the aggregation and differentiation of multifunctional stem cells,while promoting the expression of downstream genes such as osterix,alpl,bgp,and promoting the differentiation and ossification of osteoblasts.Afterwards,the expression of genes related to intermuscular bones mineralization and sclerosis,such as alpl,bgp,fgf23,and sost,produces phosphate to initiate bone mineralization,collagen fiber mineralization,and osteocalcin attachment,which promote the final formation of intermuscular bones. |
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