Study On The Role Of Olfaction In Herbivorous Feeding Habit Of Blunt Snout Bream

Olfaction is one of the most important chemoreceptors in fish,which can widely perceive food odor and various pheromones.It plays an important role in fish feeding selection,migration and avoiding predators.Megalobrama amblycephala is a native herbivorous economic fish in China.It mainly eats all kinds of aquatic plants such as Hydrilla verticillata,Myriophyllum spicatum and Spirodela polyrrhiza.In order to explore the role of olfaction in the herbivorous feeding habit of blunt snout bream,we plan to:(1)study the development of olfactory organs and the changes in the expression patterns of representative olfactory receptors(ORs)using morphology,histology and molecular biological technique;(2)explore the electrophysiological and behavioral changes of M.amblycephala after different food odors stimulation based on two food odor-induced models;(3)study the distribution of olfactory neurons and the types of olfactory neurons that can be activated after food odors stimulation through immunological methods.The results of this study will lay a foundation for further exploring the potential mechanism of olfaction and ORs in the regulation of fish feeding habits,deepen the cognition of fish olfactory recognition,and provide theoretical basis for the development of efficient food attractants.The main findings are as follows:1.Study on of olfactory organs development and gene expression patterns of representative olfactory receptor genes in blunt snout breamThe olfactory organs development of blunt snout bream was investigated using histological and morphological methods.The results revealed that the olfactory organs of blunt snout bream were positioned on the back and in front of the eyes.The olfactory epithelium(OE)was oval-shaped near the base of the olfactory cavity.The olfactory placodes were parallelly arranged in the cranio-caudal direction.There were concave marks on the olfactory fossa’s periphery when blunt snout bream was hatching out.Afterwards,an elongated olfactory placode was formed in the olfactory fossa’s core.The arrangement of the primary olfactory placode of the unilateral OE loosens and tightens as the blunt snout bream grows and develops,and the height and number of the olfactory placode,as well as the overall surface area of the OE gradually expand and stabilize in the adult stage.q RTPCR were used to study the expression patterns of representative ORs,The findings showed that ORs expression differed in tissues from different stages of blunt snout bream development.Beta-2,9,10,and 11 were highly expressed during several phases of embryonic development,including the blastocyst stage,blastopore closure stage and olfactory plate stage(P<0.01).Beta-2,9,10,11 and Epsilon-7 were poorly expressed at from 3 dpf to 15 dpf,in the larval and juvenile stages,but highly expressed at from 30 dpf to 60 dpf(P<0.01).Beta-2,10,11 and Epsilon-10,13 were strongly expressed in the OE of 4 periods from juvenile stage to adult fish stage(including 3-month-old,6-month-old,1-year-old and 2-year-old)(P<0.05).Beta-2,9,10,11 and Epsilon-7,10,13 were highly expressed in the OB(olfactory bulb)of 3-month-old blunt snout bream(P<0.01),but not expressed in 6-month-old to 2-year-old stage,and Beta-2,11 expression levels were low in the brain of 4 periods(P<0.05).2.Effects of food odor stimulation on olfactory electrophysiology and behavior in blunt snout breamThe electrophysiological and behavioral studies of sensitivity and preference to diverse food odors were investigated using the Electro-olfactogram(EOG)device and behavioral research device.The amplitude of the blunt snout bream’s reaction to the amino acid mixture was greater than that of Hydrilla verticillate extract.The response characteristics were similar and showed a clear dose-response relationship.The induction threshold value of blunt snout bream to amino acid mixture was 10-10.75 mol/L according to the linear regression equation,while the induction threshold value of H.verticillata extract was 10-5×.The results of direct behavioral studies showed that when the blunt snout bream was stimulated by 10-5 mol/L amino acid mixture and 0.005× H.verticillata extract,which had a significant preference for food odors.Their trajectories in the odor area were significantly increased compared to control group(P<0.05).The odor preference index of the two food odors was much lower in the blunt snout bream with the OE removed compared to control with OE(P<0.05).The blunt snout bream exhibited a strong preference for 0.005× H.verticillata extract compared to the 10-5 mol/L amino acid mixture in the labyrinth behavioral study(P<0.05).3.Effects of food odor stimulation on the expression of olfactory neurons and representative ORs in blunt snout breamWestern Blot was used to verify the expression of antibodies for clarifing the distribution of olfactory neurons in the OE of blunt snout bream’s olfactory substrate.We also investigated the types of olfactory neurons that can be activated after food odors stimulation and the expression pattern changes of representative ORs.The findings revealed that three types of antibodies(Gαs/olf,Gαo and Gαq/11)could bind to the three Gα proteins of blunt snout bream.Gαs/olf had a double band at 42-45 k Da.Gαq had a single band at 41 k Da,while Gαo had a single band at 40 k Da in the OE and brain tissue.The immunohistochemistry was used to study the distribution of olfactory neurons.The results revealed that different Gα antibodies marked different olfactory neurons.The Gαs/olf could mark the ciliated neurons at the bottom of the olfactory placode.Gαo could label the microvillus neurons in the middle of the olfactory placode and Gαq with the crypt neurons near the top of the olfactory placode.Based on p ERK immunofluorescence,the results showed that after stimulation with 10-5 mol/L amino acid mixture and 0.005× H.verticillate extract,a large number of ciliated neurons located at the bottom of the olfactory placode were activated,whereas only a small number of microvillus neurons were initiated in the control group;We further detected the changes in the expression pattern of the representative ORs of blunt snout bream after different food odor stimulations using q RTPCR.The results showed that the expression levels of OR Beta-9,10,11 and Epsilon-6,7 in the brain were significantly increased(P<0.01)after stimulated with 10-5 mol/L amino acid mixture,while Epsilon-7 was significantly increased in the olfactory bulb compared to the control group(P<0.01);Beta-2,10 and Epsilon-7 in the brain were significantly increased(P<0.01)after the stimulation of 0.005× H.verticillata extract,while Epsilon-7 was significantly increased in the OB(P<0.01).