| The olfactory sac of fish is an important sensory organ which plays an important role in foraging,species identification,reproduction and migration.Mandarin fish(Siniperca chuatsi)is a typical carnivorous fish that feeds on live bait for life.Because Mandarin fish refuses to eat dead bait,it is speculated that its olfaction plays an important role in its living bait feeding habits.In this paper,paraffin sections,HE staining,and Massorn staining were used to observe the histological features and early development of olfactory sac in adult fish and early developmental stages(3-55 days after hatching).The ultrastructure of olfactory epithelium cells was observed by immunohistochemistry,scanning electron microscopy and transmission electron microscopy.The gene structure and characteristics of TAAR13c-like were preliminarily studied.The tissue expression of TAAR13c-like and the expression changes stimulated by cadaverine and putrescine were detected by fluorescence quantitative PCR,which provided basic data for olfactory structure and olfactory recognition of Siniperca chuatsi.The results are as followings :(1)In order to understand the structure and early development characteristics of olfactory sac of mandarin fish,samples of mandarin fish aged 3-55 days were collected.The morphological structure of olfactory organ,structure and early development of olfactory epithelium were observed by paraffin section,HE staining,Massorn staining,immunohistochemistry,scanning and transmission electron microscopy.The results showed that Siniperca chuatsi had two pairs of nostrils,and the anterior and posterior nostrils were closely linked,with flaps.The olfactory sac is located in the olfactory cavity,which is composed of 16-20 primary olfactory plates.It is a G-type olfactory sac,and the primary olfactory plate generates secondary olfactory plates through folds.The distal edge of olfactory plate is a non-sensory area,and the sensory area is mainly located at the middle and proximal ends of olfactory plate.Under the scanning electron microscope,there were a large number of cilia coverings on the surface of sensory area,and microvilli on the surface of secondary olfactory plate.There are convex ridges on the surface of the non-sensory region.Olfactory epithelial cells can be divided into six categories: cilia non-sensing cells,cilia sensing cells,microvilli sensing cells,supporting cells,basal cells and mucous cells.From larvae to young fish,the olfactory substrate of 1-7 days old larvae was thin and the cells were loosely arranged.At 7 days of age,the olfactory base plate was thickened to form an olfactory fossa with cilia on the surface of olfactory epithelium.At 10 days of age,the olfactory base continued to sink into the olfactory fossa and formed an olfactory cavity;at 26 days old,the mucosa consisting of olfactory epithelium and lamina propria uplifted from the base of olfactory sac to form the first pair of primary olfactory plates;four pairs of primary olfactory plates were formed in 35 days old young fish;eight pairs of primary olfactory plates were formed in 55 days old young fish.Siniperca chuatsi early olfactory sac development is slow,primary olfactory plate lateral parallel arrangement,has not formed secondary olfactory plate.(2)In order to explore the correlation between live prey and trace amine receptors in Siniperca chuatsi,TAAR13c-like sequence was obtained from its genome sequence.The expression of TAAR13c-like gene in the olfactory sac,oropharyngeal cavity,skin,gill,and brain was detected by real-time quantitative PCR,and the expression changes of TAAR13c-like in the above tissues were detected under the stimulation of different concentrations of cadaverine and putrescine.The results showed that the total length of mandarin fish TAAR13c-like DNA was 1370 bp,including one intron and two exons.The intron length is 231 bp,the first exon length is127 bp,the second exon length is 1012 bp.This sequence encodes 308 amino acids with14 protein binding sites and forms seven hydrophobic α-helix transmembrane structures.TAAR13c-like was mainly expressed in the olfactory sac and the oropharyngeal cavity,followed by the gill,brain and skin.At the concentrations of 1μM,10 μM putrescine and cadaverine,the expression of TAAR13c-like in the olfactory sac,oropharyngeal cavity and brain was significantly lower than that in the control group,while at the concentrations of 100 μM and 1000 μM putrescine and cadaverine,the expression of TAAR13c-like in the olfactory sac and oropharyngeal cavity was significantly higher than that in the control group,and there was no significant difference in the expression of TAAR13c-like in the brain.At 1 μM putrescine and cadaverine concentrations,the expression of TAAR13c-like in skin and gill was significantly lower than that in the control group.At 10 μM,100 μM putrescine and cadaverine concentrations,there was no significant difference in the expression of TAAR13c-like in skin and gill,but at 1000 μM cadaverine and putrescine concentrations,the expression was significantly higher than that in the control group.In conclusion,the mandarin fish olfactory sac is the main expression site of TAAR13c-like,and it is speculated that the mandarin fish TAAR13c-like gene has a certain relationship with its live bait preference. |
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