Early Development Of Taste Buds And Pseudogenes Of Putative Sweet Taste Receptors (T1R2s) Of Mandarin Fish (Siniperca Chuatsi)

Mandarin fish(Siniperca chuatsi)is one of the typically carnivorous fish,which eat live fish and shrimp for all their life.The predation behavior of mandarin fish mainly depends on visual and lateral line systems,the sense of smell and taste are also involved in the process of food recognition.In this paper,histological characters and early development of taste buds of mandarin fish at 3-28 days old were observed through the paraffin section and H.E.staining,the sub-types and density of taste buds at 28 days old were observed via scanning electron microscopy.The full length c DNA sequences of the sweet taste receptor(T1R2)gene was cloned,the development and tissue expression of T1R1 and T1R2 and T1R3 were compared,and tissue distribution of T1R1,T1R2 and T1R3 in oropharyngeal cavity were also observed by immunohistochemical study.The main results are as follows:(1)In order to understand histological characters and early development of taste bud of mandarin fish,the sample of 3,7,14,21,28 days old were collected and observed through the paraffin section,H.E.staining,and further observed via scanning electron microscopy.The results showed that the mouth was not open at 3 days old,the taste bud is undifferentiated;at 7 days old,the mouth was opened,taste bud was in oval shape with gentle projection;at 14 days old,lower jaw began to develop,and taste bud was conical,meanwhile,the protrusion height was increased;at 21 days old,taste buds was in trapezoidal shape with obvious projection,at 28 days old,taste bud developed completely.Taste buds has three main types were observed in scanning electron microscopy: type I taste buds was in ball-like shape with contained quantities of microvilli,its processes were higher than the mucosa epithelial,taste pores bumped out;type II taste buds contained a few microvilli,its processes slightly higher than the mucosa epithelial,taste pore concave beneath;type III taste buds contained a little microvilli,its processes almost coplanar with the mucosa epithelial and the smooth inner hole.Type I taste buds was maximum,type II was minimum,in the jaw,pharynx,while large amounts of type I was in the tongue without type III.The results showed that taste bud development in the early stage played an essential role in feeding,type I and II taste buds were properly the main types in mechanical and chemical recognition during food intake.(2)To testify whether mandarin fish exhibit various feeding behaviors to different taste foods,the experiment fish is divided into umami group,sweet group and control group,the predation time,predation rate and the swallowing rate were observed and compared.The results showed that there is no difference between the umami group,the sweet group and the control group in predation time and predation rate,however,the preys were swallowed in the umami group and the control group,whereas the preys were disgorged after predation in sweet group.The swallow rate in the umami group is18.6 times higher than that of the sweet group.This may indicate that mandarin fish has an obvious preference for umami food.(3)The full-length c DNA sequences of the sweet-taste receptor gene(T1R2)of mandarin fish was cloned using RACE(Rapid Amplification of c DNA Ends),the developmental expression of T1R1,T1R2 and T1R3 genes(3,7,14,21,28 days old)and tissue expression in oropharyngeal cavity were analyzed by real time PCR,and tissue expression of T1R1,T1R2 and T1R3 were also detected by immunohistochemistry.The results show that the T1R2 genes include two copies(T1R2a1,T1R2a2),the T1R2a1 c DNA consists of 1791 bp nucleotides,including a 271 bp 5’ untranslated region(UTR),a 713 bp 3’ UTR,a 807 bp open reading frame(ORF),it encoded 269amino-acid residues with seven transmembrane domain structures,without containing signal peptides.The T1R2a2 c DNA consists of 1049 bp nucleotides,including a 271 bp5’ UTR,a 322 bp 3’ UTR,a 456 bp ORF,it encoded 152 amino-acid residues with 4transmembrane domain structures,without containing signal peptides.Compared with the functional areas of T1R2 in other fish species,T1R2a1 has only one complete functional area,whereas T1R2a2 has no complete functional area.It is speculated that T1R2a1 gene is a putative functional gene and T1R2a2 is a pseudogene.At 3 days old,expression of T1R1,T1R2 and T1R3 genes were observed in small amounts,at 7 days old,the expression levels of T1 Rs significantly increased(P<0.05),expression of T1R1 and T1R3 were 3.06 times and 1.47 times of the T1R2 expression,at 28 days old,the expression of T1 Rs gene tended to be stable.T1R1 and T1R3 are mainly expressed in the tongue and pharynx,they also expressed in the upper jaw and the lower jaw,the lowest expression is in the gill,T1R2 was mainly expressed in the tongue and the pharynx,less in the upper jaw and the lower jaw,but not expressed in the gill.Immunohistochemistry revealed that T1R1 and T1R3 were mainly distributed in the tongue and pharynx,the upper and lower jaw and the gill,but positive reaction of T1R2 is not detected in oropharyngeal cavity.It is speculated that pseudogenization of T1R2 in mandarin fish may be associated with their carnivorous feed preference.