The Identification Of New Type Of Intermuscular Bone(IB) As Well As Myosepta Development And Related Gene Expression Analysis In Represent Fish Species With Or Without IB

Intermuscular bone(IB),also known as intermuscular spine or intermuscular small bone,which is a small bony spine located in the bilateral muscle septum of the vertebra.Clupeiformes and Cypriniformes are the mainly economic cultured fish in the world,but species in these two superorders have a certain number of intermuscular bones.However,the existing of IBs has an adverse effect on human consumption as well as products export and further processing,which affects economic value seriously.Blunt snout bream(Megalobrama amblycephala),also known as Wuchang fish,belongs to Cypriniformes,Cyprinidae,Megalobrama.It is an important freshwater aquaculture species in China;however,the existence of IBs restricted its economic value.Nile Tilapia(Oreochromis niloticus),commonly known as African crucian carp,belongs to Perciformes,Cichlidae,which does not possess IBs(no epineural and epipleural).It is an excellent freshwater aquaculture species in south of China.M.amblycephala and O.niloticus could be used as represent fish species to study the development of IBs.At present,many studies related to IBs focused on its morphology;however,the molecular regulatory mechanism of IBs is rarely studied.To better understand the IBs morphology,we collected fish samples from the upper,middle and lower reaches of the Yangtze river,and counted the collected samples number of IBs,which covered 10 orders,129 species.The statistics showed that the morphology of IBs in Coilia nasus was different from Cyprinidae species.We studied the histology and morphology of IBs in C.nasus and found a new type of IBs.To further explore the mechanism of IB development,this study used M.amblycephala and O.niloticus as the represent fish species.Their histology structures were characterized and expression of genes related to IBs development in different stages were analyzed respectively.The mainly research results of the thesis were as followed:(1)In order to further explore the situation of IBs in freshwater fish in China,the fishes' IBs in the upper,middle and lower reaches of the Yangtze river was calculated.Among the 129 species collected fishes,the epineurals and epipleurals were found in the Cypiformes and Clupeiformes.The number of IBs ranged from 86 to 180 in Cypiformes fish,while Clupeiformes fish had more IBs.In addition,a new type of IBs was found in Coilia nasus and C.brachygnathus.To study the characteristics of the new type of IBs,we documented the number,morphology,and distribution of IBs in C.nasus.The morphology of IBs in C.nasus was not different with Cyprinidae species;however,the distribution of IBs was quite different.Besides epineurals,epicentrals,and epipleurals,we found two others categories of IBs in C.nasus,which are located in the dorsal and ventral parts on both sides of the vertebrae.According to the reference,we called them dorsal and ventral myorhabdoi,respectively.These types of IBs were also identified in C.brachygnathus.The morphology of these IBs showed non-forked type(“1”or“(”).The number of IBs in C.nasus ranged from 492 to 543,and the number of dorsal myorhabdoi,epineurals,epicentrals,epipleurals and ventral myorhabdoi ranged from 92 to 135((?)=114),114 to 142((?)=133),28 to 51((?)=42),138 to 153((?)=142),66 to 98((?)=89),respectively.IBs can be stained by alizarin red,but not alcian blue.For epineurals and epipleurals,the IBs were connected with one by one through connective tissues.(2)We compared the myosepta development based on histological analysis between M.amblycephala and O.niloticus,in order to reveal the possible tendon differentiation differences between of species with or without IBs.The histological results showed that dying condensation in the myosepta at 20 day post hatch(dph)in M.amblycephala,then formed all the IBs without the cartilaginous phase until 50 dph based on alizarin red S,alcian blue-nuclear red,HE,toluidine blue staining methods.However,in O.niloticus,the dying condensation was not observed in the myosepta.(3)To further explore the molecular mechanism of IBs,several genes(entpd5,phex,bgp,alpl,sp7,runx2,fgf23,col1a2,scx)related to bone development were selected out and analyzed.Phylogenetic analysis of these genes indicated that fish species with IBs and fish species without IBs formed separate clusters,respectively.The different genes decomposed into separate group,indicating the function of these genes has been conserved throughout evolution.In order to study the possible function of these genes in the muscle for IBs formation,we compared their expression levels in M.amblycephala and O.niloticus at different stages(5,10,15,20,25,30,40,50,60 dph and one-year old fish).Compared with O.niloticus,the expression of all the genes in M.amblycephala were increased at 20 dph.Together with the histological results,we speculated that 20dph is an important stage for IB formation.Meanwhile,the expression of sp7 and entpd5a reached the highest level at 40 dph,bgp at 60 dph,suggesting that they are potential involvement in IB mineralization in M.amblycephala.However,the relative expression level of alpl was decreased at all the stages in O.niloticus,while bgp and entpd5a showing little changes,fgf23 having an increase trend,especially at one-year old fish.The results of this study suggest that these genes play the roles as inducer(sp7,entpd5a,alpl and bgp genes)or inhibitor(fgf23 gene)along with IBs'ossification and mineralization.