| Anemone fish belongs to Perciformes,Pomacentridae,Amphiprion.As a tropical seawater ornamental fish,anemone fish is widely loved by people for its diverse body colors.Among them,Amphiprion percula and A.ocellaris have three white vertical stripes on the body surface of the adult fish,which are located behind the eyes,in the center of the dorsal fin and at the tail,respectively.The surface of the body is orange,black and white areas in sequence,but the black areas next to the white vertical stripes are wider in the former than in the latter.When the white vertical stripes in the center of the dorsal fin of the artificially selected A.percula developed into cross-shaped stripes,a new variety was obtained:Picasso clownfish.The adult A.frenatus has a slightly different body color than the former two anemone fishes,with a bright orange-red color throughout and only a white stripe behind the eye.However,the color of the artificially farmed anemone fish is lighter than that of the wild anemone fish,and the characters of the offspring of hybrid breeding are changeable,which affects the economic benefits.Therefore,this paper mainly takes A.frenatus（wild type）,A.ocellaris（wild type）and A.percula（artificially selected and cultivated）as the research objects,combining body color changes during early development of anemone fish with molecular biotechnology to lay a foundation for the follow-up research on the mechanism of body color in the anemone fish.The results are as follows:1、Observation and study on the reproductive habits of different fishes of AmphiprionArtificially reared A.frenatus（wild type）,A.ocellaris（wild type）and A.percula（artificially selected and cultivated）in the laboratory to observe their reproductive habits.After the above-mentioned fish spawned and successfully fertilized,the embryos are taken at the cleavage stage,somite stage,turnover period,vascularization,organ formation stage and hatching stage,then observed and recorded using microscope（Nikon）.It was observed that the embryos of A.frenatus were orange-red,and theembryos of A.ocellaris（wild type）and A.percula（artificially selected and cultivated）were orange-yellow.Melanocytes appear as punctate,which are begin to appear during the somite stage（33 hours post fertilization,hpf）,and although in small numbers,are scattered throughout the embryo.Erythrocytes appear later,appearing in the tail and head approximately during the organ formation stage（105 hpf）,and are smaller in volume and number than melanocytes.Xanthophore and iridophore not observed throughout embryonic development.It takes at least 8 or 9 days to develop to the hatching stage.At this time,the whole embryo appears transparent,the silvery eyes can be directly observed,and the yolk has been consumed to a minimum.2、Changes in body color of larvae and juvenile of different fishes of AmphiprionThe larva and juveniles of A.frenatus（wild type）,A.ocellaris（wild type）and A.percula（artificially selected and cultivated）were obtained by artificial breeding,and observed with a microscope.Among them,the newly hatched A.frenatus and A.ocellaris are colorless and transparent.The color gradually deepens as it grows and develops,and the stripes begin to appear about 9 or 10 days post hatching（dph）.The larvae of A.percula（artificially selected and cultivated）are orange-yellow,develop more slowly than the first two,and begin to appear stripes about the 14 dph.There are three different phenotypes in the offspring of A.percula（artificially selected and cultivated）:The body surface is basically completely white,which is called Platinum clownfish;the central vertical stripes on the dorsal fin develop into cross-shaped stripes,which is called Picasso clownfish,and the same color as the wild type is called A.percula.The offspring of A.frenatus and A.ocellaris are the same as the parent phenotype.In the case of normal development,the larvae of A.ocellaris grows from the appearance of stripes to the adults,the three white stripes on the body surface will not change,and the white stripes of A.frenatus will appear in turn from the head to the tail,as it grows and develops,the white stripes will disappear from the tail to the head in turn,and finally only one white stripe remains on the head.3、Screening of reference genes in q PCR application of anemone fishTaking the embryos and tissues of A.frenatus and A.ocellaris as samples,the expression stability ofβ-actin,18S r RNA,Ef-1αand GAPDH was analyzed by ge Norm and Norm Finder reference gene screening software.As shown by data,the largest Ct（Cycle threshold）are commonly found in the expression of Ef-1α,indicating that its expression level is low.Ge Norm analysis will get the corresponding expression stablity Value（M）,the smaller M,the higher the stability.In the adult tissues of A.frenatus and A.ocellaris,the stability arrangement was 18S r RNA=β-actin=GAPDH>Ef-1α,and in the embryonic development of A.frenatus and A.ocellaris,the stability arrangement was18S r RNA=β-actin>Ef-1α>GAPDH.Paired variation analysis values（V）are Vn/Vn+1=V2/V3=0.904>0.15,so,there are 3（n+1）candidate reference genes suitable for anemone fish gene expression studies:18S,GAPDH andβ-actin.But during embryonic development,18S andβ-actin are more stable than GAPDH.When the normalized Ct values were analyzed by Norm Finder software,it was found that in the embryonic and adult tissue samples of A.frenatus and A.ocellaris,the stability of the candidate internal reference genes was ordered as 18S r RNA>β-actin>Ef-1α>GAPDH,which was similar to the analysis results of ge Norm.The comprehensive analysis results show that the M value of 18S is the smallest,which is more suitable as a reference gene in anemone fish body color correlation gene expression analysis experiment compared with the other three reference genes.4、Expression analysis of body color related genes in early development of three species of anemone fishCombined with the changes of body color during the early development of A.frenatus（wild type）,A.ocellaris（wild type）and A.percula（artificially selected and cultivated）,the expression of related genes was analyzed by q PCR technology.The data showed that in the early developmental stages of these three species of anemone fish,foxd3,ltk,fms,soxl0,pax3,tbx2b,ednrb,dct,tyr and aim1 genes were all expressed,and may also be involved in regulating other functions of the organism.During embryonic development,melanocytes first appeared in the somite stage,at which time the expression of tyr gene significantly increased to the highest（P<0.05）,and participated in the synthesis of early melanin.During the vascularization stage of embryonic development,the expression of fms gene is increased,and then erythrocytes were observed during organ formation stage,which indicated that the differentiation sources of both erythrocytes and xanthophore may be the same.When stripes appeared,the number of iridophore increased sharply,and the proportion of melanocytes,xanthophore and erythrocytes decreased,which may be the reason why the expression level of fms gene decreased gradually during the later development of anemone fish.In the process of stripes fading,the expression of tbx2b gene continued to increase,which changed the composition and proportion of pigment cells in the center of the dorsal fin and at the tail,thus causing the disappearance of stripes in A.frenatus. |
