Cloning And Expression Of Pv5-HT₇ And Pv5-HT_2A Receptor Genes In Pseudo-black Spiny Ants And Their Effects On The Growth And Development Of Ants

5-hydroxytryptamine(5-HT),also known as serotonin,is one of the oldest biogenic monoamine neurotransmitters.5-HT was found in diverse species from nematodes and fruit flies to mammals.5-HT modulates a great variety of important physiological and behavioral functions such as reproduction,sleep,appetite,learning,pain perception,circadian rhythms,mood,aggressive behavior,feeding,energy balance,food intake,gastrointestinal and endocrine function,and cardiovascular physiology.Impaired secretion of human 5-HT have been implicated in many diseases such as schizophrenia,migraine,depression,infantile autism,obsessive-compulsive disorder,eating disorders and suicidal behavior.In insects,5-HT participates in the regulation of circadian rhythms,learning and memory,aggregation,secretion,development,aggression and heart rating.5-HT mediates specific effects by interacting with various membrane-bound receptors.To date fourteen 5-HT subtype receptors have been cloned and identified from vertebrates and invertebrates.These receptors can be divided into seven families(5-HT1-5-HT7)according to their second messenger coupling,pharmacological properties and sequence homology.Except that 5-HT3 receptor is a ligand-gated ion channel receptor,all other 5-HT receptors belong to G protein coupled receptors superfamily.In insects,four 5-HT receptors including 5-HT1A,5-HT1B,5-HT2A,and 5-HT7 were identified,and 5-HT2B is only found in Drosophila melanogaster.However,the effect of 5-HT receptor on the growth and development of Polyrhachis vicina Roger has not been reported.Polyrhachis vicina Roger is a typical eusocial insect with the caste differentiation and belongs to the genus Polyrhachis(Hymenoptera:Formicidae).P.vicina has a clear social division,complex behavioral activities and advanced neural regulation.Thus,it is a good experimental material for studying the insect growth,development and behavior regulation.Based on the above reasons,we cloned the Pv5-HT7 receptor gene and Pv5-HT2A receptor gene,and detected the mRNA and protein expression level of these two genes in different developmental stages and castes.The protein expression level of the head,thorax and abdomen in different caste adults was detected quantitatively.The protein expression location in the brain and abdomen of different caste adults were detected by immunohistochemistry technique.The workers were trained by Y maze training method.The research purpose is to understand the basic structure of these two genes,the genetic relationship with other species and the effect on the growth and development of the ants.It is quite necessary to recognize the modulation mechanism of these two genes in the ant brain nervous system and reproductive system and to explore the effect on the learning and memory of the ants.The main results are summarized as follows:1.Cloning and sequence analysis of Pv5-HT7 receptor geneThe full-length cDNA sequence of Pv5-HT7 receptor(GenBank accession KF297572.1)is 3054 bp and comprises of a 5’ untranslated region(UTR)of 790 bp,a 3’UTR of 752 bp and an open reading frame(ORF)of 1512 bp encoding a polypeptide of 503 amino acids.The calculated molecular weight and isoelectric point of Pv5-HT7 receptor protein were deduced to be 55.75 kDa and 8.44 respectively.Hydrophobic analysis suggested that seven trans-membrane domains are the major sequence characteristic of the Pv5-HT7 receptor.The phylogenetic analysis revealed that Pv5-HT7 receptor was most closely related to Apis mellifera 5-HT7 receptor.Pv5-HT7 receptor protein sequence has three glycosylation sites,three protein kinase A phosphorylation sites,four protein kinase C phosphorylation sites and one palmitoylation site.All insect 5-HT7 receptor protein sequences have a common carbon terminal of the PDZ domain(Glu-Ser-Phe-Leu).Comparison of Pv5-HT7 receptor amino acid sequence with other species 5-HT7 receptors revealed high sequence similarities to insect 5-HT7 receptors.The multiple sequence alignment analysis showed that Pv5-HT7 receptor has 78%identity with A.mellifera,69%identity with B.mori,68%identity with D.melanogaster and 55%identity with P.rapae.2.Expression pattern of Pv5-HT7 receptor at the mRNA and protein levelThe Pv5-HT7 receptor mRNA was expressed in different developmental stages and castes.The Pv5-HT7 receptor mRNA expression level was obviously higher in pupae than in eggs and 1st-4th instars larval stages.And in the adults,the Pv5-HT7 receptor mRNA expression quantity was worker>male>female,which may exhibit some significance of Pv5-HT7 receptor in regulating pupae development as well as caste differentiation.The higher expression of Pv5-HT7 receptor gene in workers may be closely related to the foraging behavior of the workers.The expression of Pv5-HT7 receptor protein was analyzed in the different developmental stages and castes and individual’s body segment by Western blot.The results revealed that the protein was mainly expressed in pupae and adults stage,almost not expressed in egg and 1st-4th instar larvae stage.However,the protein expression of pupae stage was significantly lower than the adults.Among the adults,the expression of Pv5-HT7 receptor protein was the highest in the whole body of male.In the different segments of male,the highest expression was detetced in the thorax and the lowest in the abdomen.The expression of Pv5-HT7 receptor protein was intermediate in the whole body of the female,with the highest in the head of female and the lowest in the abdomen.The expression of Pv5-HT7 receptor protein was the lowest in the whole body of worker,with the lower expression in the thorax of worker,and the higher in the head and abdomen.The detection of Pv5-HT7 receptor protein was not consistent with the corresponding mRNA expression level.This situation may be due to the regulatory mechanism of the Pv5-HT7 receptor gene at the transcription and translation level,and may also reflects the specific modulation of the Pv5-HT7 receptor protein in different caste adults.The high expression of Pv5-HT7 receptor in the whole body and thorax of male may indicates that Pv5-HT7 receptor get involved in the mating flight,the development of male organs and the complexity of movement behavior.However,the expression of Pv5-HT7 receptor protein in the head of female and worker is relatively high,which may be related to learning and memory and overall coordination.The higher expression of Pv5-HT7 receptor protein in the worker abdomen may play an important role in regulating the digestive function of worker.3.Cloning and sequence analysis of Pv5-HT2A receptor geneThe full-length cDNA sequence of Pv5-HT2A(GenBank accession KJ666537.2)receptor was 2965 bp and comprises an open reading frame of 1926 bp encoding 641 amino acids,a 5’UTR of 593bp and a 3’UTR of 446 bp.The molecular weight and isoelectric point of Pv5-HT2A were inferred to be 69.68 kDa and 9.60 respectively.The phylogenetic analysis revealed that Pv5-HT2A receptor was most closely related to Pogonomyrmex barbatus 5-HT2A receptor.There are eight glycosylation sites,five protein kinase A phosphorylation sites,four protein kinase C phosphorylation sites and three palmitoylation sites in the Pv5-HT2A receptor protein sequence.Comparison of Pv5-HT2A receptor amino acid sequence with other species 5-HT2A receptors revealed high sequence homology to ant 5-HT7 receptors.The multiple sequence alignment analysis showed that Pv5-HT2A receptor has 84%identity with Pogonomyrmex barbatus and 57%identity with Drosophila melanogaster.4.Expression pattern of Pv5-HT2A receptor at the mRNA and protein levelThe expression of Pv5-HT2A receptor mRNA was detected in all the different developmental stages and castes.Moreover,the expression in eggs and 3rd instar larval and pupal stage was significantly higher than those in 1st,2nd and 4th instar larval stages.Among the adults,the highest expression was in male and lowest in female.Pv5-HT2A receptor mRNA may play a major role in the development of pupae and males,as well as the caste differentiation.Pv5-HT2A receptor protein in eggs was expressed at a high level.The protein expression in 4th instar larval stage was higher than 1st,2nd and 3rd instar larvae.The expression was the highest in males,relatively lower in females,and almost invisible in workers.The results showed that although the expression of Pv5-HT2A receptor protein was not completely consistent with that of mRNA level,the expression of Pv5-HT2A receptor protein was relatively consistent in the egg stage,but not in the 3rd and 4th instars.Therefore,Pv5-HT2A receptor plays an important role in maintaining the normal development of embryo and the critical developmental stage of 3rd and 4th instar larvae.The expression level of Pv5-HT2A receptor protein in the males was relatively consistent with that of mRNA level,and was higher than the other two adults,which reflects the important role of Pv5-HT2A receptor in regulating male marriage flight and aggressive behavior,female dominant mating behavior.The Pv5-HT2A receptor protein expression level in head was worker>male>female.It is suggested that Pv5-HT2A receptor protein participated in regulating the feeding,learning and memory of worker.In females,the Pv5-HT2A receptor protein expression was the highest in abdomen and the lowest in head,which may be related to the regulation of female reproductive abilities.In the males,the protein expression was the highest in the thorax and the lowest in the abdomen.This may be related to regulating the movement ability of male.5.Immunohistochemical study of Pv5-HT7 receptor and Pv5-HT2A receptor proteinPv5-HT7 receptor protein was strongly expressed in Kenyon cells,mushroom body and optic lobes of P.vicina brain in different castes.The expression was moderate in antennal lobes,but weak in pedunculus and α-lobes.The expression of Pv5-HT2A receptor protein was higher in the lip of mushroom body of the different castes,but weaker in the collar and basal ring.The expression of Pv5-HT7 and Pv5-HT2A receptor protein was the most in the retina and lamina,and the expression of Pv5-HT7 and Pv5-HT2A receptor was also strong in medulla and intraoptic lobula.Mushroom body is the learning and memory center in insect brain.According to the analysis of its expression,Pv5-HT7 receptor and Pv5-HT2A receptor may participate in learning and memory behavior of P.vicina.Pv5-HT7 receptor and Pv5-HT2A receptor was involved in the visual information adjustment and treatment process of P.vicina.Pv5-HT7 receptor protein and Pv5-HT2A receptor protein may be involved in the regulation of structural characteristics of brain nervous system development related to the caste differentiation in the adults.The expression of Pv5-HT7 receptor protein was strongly positive in the oocyte of the growth stage and the differentiation stage of the workers and females.Pv5-HT7 receptor protein was also expressed intensely in the oocyte of the yolk forming stage and nurse cell of the females,and in the spermatocyte and seminal vesicle of males.The positive expression of Pv5-HT2A receptor protein was weak in fat body of the workers abdomen.The positive granules of Pv5-HT2A receptor protein were strong in the growth,differentiation and yolk forming stage oocyte and nurse cell of the females,but relatively weak in fat body of the females.The positive expression of Pv5-HT2A receptor protein was strong in spermatocyte and seminal vesicle of male.The positive staining was relatively weak in the accessory glands,ejaculatory ducts and fat body of the males.It is suggested that Pv5-HT7 receptor protein and Pv5-HT2A receptor protein play a major role in regulating the development of the male and female germ cells.Pv5-HT7 receptor protein and Pv5-HT2A receptor protein may be involved in the regulation of the development and structural characteristics of the abdomen reproductive system related to the caste differentiation in the adults.6.The effect of Pv5-HT7 receptor and Pv5-HT2A receptor gene expression by Y-maze training.The ants were trained by using Y-maze.As the number of experiments increased,the time for the workers to find the target was shorter.The relative expression levels of Pv5-HT7 receptor and Pv5-HT2A receptor in untrained and trained workers were detected.The results demonstrated that the relative expression levels of Pv5-HT7 receptor and Pv5-HT2A receptor increased after behavioral training.This may revealed that behavioral training stimulates the increase of 5-HT level in the brain of workers and induces the expression of Pv-HT7 and Pv5-HT2A receptor,which indicates that Pv5-HT7 receptor and Pv5-HT2A receptor are closely related to the learning and memory behavior of P.vicina.In this study,the full-length cDNA sequences of Pv5-HT7 receptor and Pv5-HT2A receptor genes were obtained for the first time.Phylogenetic analysis showed that Pv5-HT7 receptor gene and Pv5-HT2A receptor gene were closely related to those of the other ant species or species of the order Hymenoptera.The results of real-time PCR and Western blot showed that Pv5-HT7 receptor and Pv5-HT2A receptor in mRNA and protein levels were expressed in the different developmental stages and castes,which reflects that these two receptors play a major role in regulating the development and caste differentiation of P.vicina.Because the mRNA and protein levels of these two genes expressed in the different developmental stages and castes have some differences,which showed that both genes play a certain regulatory role in the specific development,caste differentiation and the specific function of different caste adults.The present study has scientific significance to further understanding of the Pv5-HT7 receptor gene and Pv5-HT2A receptor gene,regulating the growth and development and caste differentiation of the social insect ants.This study lays a good foundation for further research of Pv5-HT7 receptor gene and Pv5-HT2A receptor gene for the development of higher animals and even human beings disease treatment.