Study On Development Of Gills And Gene Expression Of Eda And Edar In Mandarin Fish(Siniperca Chuatsi) And Silver Carp(Hypophthalmichthys Molitrix)

Mandarin fish(Siniperca chuatsi),commonly known as osmanthus fish,belongs to Perciformes Percoidea and is a precious economic fresh water species with specific food preference,only accepting live prey.While silver carp(Hypophthalmichthys molitrix)feeds on plankton.The long,dense and soft gill rakers of silver carp are quite different from mandarin fish's short,sparse and tough ones.Since gill rakers plays an important role in filter-feeding,the difference of gill rakers between these two kinds of fish might be a main cause of feeding differentiation.Fish in early development of 1-7 days post-hatch(dph)for mandarin fish and 4-10 dph for silver carp were fixed with 10%formalin and 4%paraformaldehyde.The samples treated with 10%formalin were used for alizarin red staining,and then the gill rakers were observed under the microscope.To further understand the genetic factors affecting gill development,gene structure of eda and edar were analyzed between fish species.The number of eda extrons and introns was conserved in fishes,while the number of edar extrons varied between species,suggesting that the structural differentiation of edar might cause difference in the development of gill arches and gill rakers.Synteny analysis of eda and edar showed that several genes upstream were also related to the development of integumentary appendages.The close relationship of location may indicate their functional concordance.The samples treated with 4%paraformaldehyde were for in-situ hybridization to measure the expression of edar genes in seedlings of silver carp.In comparison,the expression of genes in adult fish was measured by RT-PCR.All conclusions are as follows:1.The alizarin red staining(ARS)method was used in morphologic observation of gill rakers in early stage.The number of gill arches in seedlings was the same as that of adult fish,while gill arches become longer and thicker during the development.The gill rakers are born linearly on the gill arch both in silver carp and in mandarin fish.The number of gill rakers of silver carp seedlings increased from 8 to 13 at 4-10 dph,and in adult fish gill rakers are closely connected into a thick menbrame with no spacing between them.The average spacing between gill rakers decreased from 495.39 ?m to 387.38 ?m In mandarin fish,the number of gill rakers didn't change much,at 1-6 dph,gill raker number increased from 6 to 7 and the spacing decreased,at 7 dph,the spacing increased to 475.35-749.55 ?m,and the number of gill rakers were still 8.The gill rakers are dotted in gill arches and difficult to be observed.In adult mandarin fish,8 gill rakers were observed and they become longer and tougher.2.The result of phylogenetic tree analysis implied relevance of mandarin fish to Pagothenia borchgrevinki,Larimichthys crocea,Gasterosteus aculeatus and Oreochromis niloticus in the development of integumentary appendages.3.Specific primers of eda and edar were designed and successfully screened based on sequences from transcriptom library of mandarin fish and genome of silver carp.RT-PCR was performed to analyze tissue expression of eda and edar.Eda was highly expressed in liver,gill filament,gill arch and skin of silver carp,and in gill arch,gill rakers and skin of mandarin fish.While edar was highly expressed in gill arch,gill rakers and skin both in mandarin fish and silver carp.The egression of eda and edar was different in silver carp,but the same in mandarin fish,which may be the reason for different development patterns of silver carp gills and mandarin fish gills.4.On basis of the RT-PCR result and the alizarin red staining result,silver carp,which has much difference in its gill rakers development during the early stage,was selected to do research on relationship of gene expression and gill development.Specific primers of RNA probe used by in situ hybridization(ISH)were designed and successfully screened,and the expressionof edar in early development(13-22 dph)of silver carp was performed.Edar was expressed in gills and the color of ISH became increasingly obvious with the development.For edar was highly expressed in gill arch and gill rakers of adult silver carp,the increasingly high expression of edar in seedlings may indicate the development of gill arch and gill rakers in the early stage.This result conforms to the morphology observation by alizarin red staining and this change adapts to filter-feeding of silver carp.