| Like other animals,the insect's brain is the center that controls behavior.It receives sensory information from the environment,and recognizes,encodes,stores and processes the information,and finally sends out instructions to guide insects to produce behavioral responses.Adapting to the complex sensory environment,insect brains have evolved to be highly regionalized.Different regions process different information,for example,the mushroom body is the learning and memory center.At the same time,the brain also contains a large number of neurons and glial cells,and through the release of different types of neurotransmitters or neuromodulation to regulate various physiological processes and behaviors.All these perspectives provide ample opportunities to study the functions of the nervous systems in general.Additionally,neuroethological studies have shown that the insect nervous systems can be a useful target of research aiming at developing bio-active chemicals that can regulate insect behaviors,by unraveling the coding and regulatory mechanisms of environmental stimuli.The dogma of structure-function relation determines that before studying the brain function,we need to conduct detailed research on its structural characteristics.This principle guides my entire thesis on the oriental fruit fly Bactrocera dorsalis(Hendel),which is one of the most destructive pests of fruits and vegetables around the world,causing huge economic losses each year.As a polyphagous insect,it can use about 250 species of plants as host,including mango,guava,peach,etc.That olfaction plays an important role in the host search of oriental fruit fly inspires many scientists to develop corresponding behavioral regulators based on olfaction.Studies of brain structure and olfactory pathways of oriental fruit fly are promising in developing environment-friendly efficient control technologies by revealing neural regulation mechanisms underlying host selection of this pest.In this study,immunohistochemical staining,laser confocal imaging,3D reconstruction,and neuron trace techniques were used to study the anatomical structure of the brain of B.dorsalis,and the 3D model of brain structure was constructed.Furthermore,the size,composition and spatial location of each neuropil in the brain were analyzed,especially for the primary olfactory central-the antennal lobe(AL),we count the number,analyze the volume and sexual dimorphism of each glomerulus.The projection patterns of antennae and maxillary palp sensory neurons in the nervous system and the distribution patterns of two biogenic amines in the brain neuropils were studied.The main findings are as follows:1.Three-dimensional reconstruction and structural characteristics of brain neuropils of B.dorsalisFirst,the synapsin antibody can be used to label the neuropils regions of the brain;In B.dorsalis,the cerebral ganglia(CRG)and gnathal ganglion are fused,with a hole in the middle of the esophagus bypassing.According to the insect brain naming system,the brain can be divided into three structural levels,namely,supercategories neuropil,neuropil and neuropil subregion.A total of 12 kinds of supercategories neuropils were identified in the whole brain,among them,the boundaries of the optic lobe,antennal lobe,mushroom body,central complex,and lateral accessory lobe complex were relatively clear and easy to identify.Among the five supercategories neuropils,the optic lobe accounted for the highest proportion in the whole brain,59.64-60.75%,followed by antennal lobe(1.82%).In addition to the above-mentioned supercategories neuropils,the brain also contains other neuropils with blurred boundaries,accounting for 34.63%of the female and 35.41%of the male in the whole brain.They are the superior neuropils(SNP),the ventromedial neuropils(VMNP),the inferior neuropils(INP),the ventrolateral neuropils(VLNP),periesophageal neuropils(PENP),gnathal ganglia(GNG)and lateral horn(LH).The 12 kinds of supercategories neuropils in the brain can also be further divided into 43 neuropils,in which the medulla,lateral accessory lobe,fan-shaped body,and protocerebral bridge can also be further divided into multiple neuropils subregions.The anatomical organization of the brain is similar to other insects,but the proportion of each neuropil is not the same.2.Structural characteristics of antennal lobe and its relationship with the higher olfactory center in B.dorsalisIn this chapter,the morphological characteristics of antennal lobe(AL),including the number and volume of glomeruli,were studied in detail by means of immunostaining and laser confocal imaging techniques.In addition,each glomerulus was identified and named.The AL of the oriental fruit fly is located in the deutocerebrum,the anterior part of which is connected to the antennal nerve,and the posterior side is adjacent to the lateral accessory lobe,it contains 64-65 glomeruli,which are layered around the AL.The volume of the glomeruli ranges from 806 to 19592?m~3,and the largest is the PV1.The overall glomerular volume of the two sexes is not statistically different.However,eight glomeruli are sexually dimorphic:four(named AM2,C1,L2,and L3)are larger in males,and four are larger in females(A3,AD1,DM3,and M1),indicates a differentiation in the olfactory perception of odors.It contains three typical medial(m ALT),media lateral(ml ALT),and lateral(l ALT)antennal-lobe tracts form parallel connections between the AL and protocerebrum.In addition to these three tracts,we also found a transverse antennal-lobe tract(t ALT).l ALT and ml ALT projecting directly to the LH,and m ALT first projects to the ipsilateral calyx(CA),and then terminates at the lateral horn.The m ALT divided into two roots:the thicker dorsal root(DR)and the thinner ventral root(VR)to innervate the AL.The somata location of the PNs whose axons form the DR and VR are different.3.Central projections of antennal and maxillary palp nerves in the central nervous system of the B.dorsalisAfter an overall understanding of the structure of the brain neuropils,the projection area of the antennal and maxillary palp nerves in the brain was studied by using tracer backfill,immunostaining,and laser confocal imaging techniques.The aim of this study was to clarify the projection pathway of these two sensory organs of this fly.The axons of the maxillary palp sensory neurons project to the gnathal ganglion(GNG)and the AL,while the antennal sensory neurons mainly project to the AL,dorsal protocerebrum,the antennal motor and mechanosensory center(AMMC),GNG and the thoracico-abdominal ganglion(TAG).The AL is the most important projection area,indicates that olfactory sensation is an important function of antennae and maxillary palp.From the backfill experiments of different segments of the antennae,we can see that the flagellum nerves of the antennae project into the AL,while the pedicel mainly projects to AMMC.The projections of neurons within the arista to the two glomeruli VP2 and VP3 on the posterior side of the AL,furthermore,there are two bundles of arista axons projected to the superior neuropils(SNP).These results suggest that different sensory neurons have different projection areas in the brain,the flagellum plays an olfactory sensory function,and they axons are mainly projected into the dorsal and lateral lobes of the AL,while the pedicel is mechanical sensory function and innervate 5 different regions of the AMMC.A bundle of nerve axons from the pedicel not only innervates the GNG,but its axons eventually project to the TAG.There is no overlap between antenna linked and palp-linked glomeruli,suggesting that antenna and maxillary palp may supply parallel streams of olfactory information to AL.In AL,56 glomeruli innervated by antennal nerve,accounting for 86.46-86.82%of the whole al.The maxillary palp nerve innervates 9 spheres of the ventral part of the AL,accounting for 13.2-13.4%.4.Distribution of biogenic aminergic neurons in the brain of B.dorsalisIn order to understand the regulation mechanism of biogenic amine on olfactory behavior,we used serotonin and tyrosine hydroxylase antibodies to label two kinds of bioaminergic neurons in the brain neuropils of B.dorsalis,respectively.The number of cell bodies and the innervation patterns of different neuropils was analyzed.There are about 86 serotonergic neurons and 339 dopaminergic neurons in the brain and their neural processes are widely distributed in the brain,indicating that they are involved in a variety of physiological and behavioral regulation.Serotonergic neural fibers were mainly distributed in the lobes,pedunculus and accessory calyx(ACA)of the mushroom body,in which the ACA was innervated by neural fibers from the ALP cell group,and the calyx and lateral horn lacked the distribution.In the AL,each glomerulus showed serotonin immunoreactivity,and it was innervated by the contralateral neuron AMP,which indicated that the regulation of serotonin on olfactory sensation was mainly at the AL level.In the brain,5HT-immunoreactive neuropils also included optic lobe,anterior optic tubercle,central complex,lateral accessory lobe,superior neuropils,inferior neuropils,and ventrolateral neuropils,but no distribution was found in AMMC and protocerebral bridge.The lobes,CA and ACA of the mushroom body contain dopaminergic neural fibers,in which the vertical lobe of the mushroom body is innervated by the PPL1 cell group,the CA and ACA of the mushroom body are innervated by PPL2ab cell body group.No distribution was found in the AL and AMMC,this suggests that dopamine mainly acts on the higher olfactory center,the mushroom body,rather than the primary olfactory center,the antennal lobe.The above results are of great significance for the future study of neuropils function and the mechanism of odor recognition and coding in AL of B.dorsalis.In future applications,antennal lobe can be used as a detector to find olfactory-based behavior regulators.In addition,the distribution data of biogenic amines in the brain provided by this study are the basis of understanding the behavioral mechanism of biogenic amines regulating olfactory perception,learning,and memory of this fly. |
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