| Chinese mitten crab?Eriocheir sinensis?is an important aquaculture species in China,and the agonistic behavior is a normal phenomenon in pond culture.It often causes limb disability and consumes a lot of energy,which is harmful for the growth and survival of commercially important crustaceans.In this study,we mainly focused on the fighting behavior of the crab,recorded the agonistic behavior of the crab within1 hour using a high-definition camera.Furthermore,we preliminarily evaluated the changes of 5-hydroxytryptamine?5-HT?or dopamine?DA?levels before and after the fight,detected the expression of downstream related receptors and some energy metabolism indicators.According to the analysis results,we first divided the agonistic behavior into three types/stages:approach,contact and fight.We found that the number and cumulative time of fight of the male-male group was significantly higher than the female-female group or female-male group?P<0.05?.After 1 hour,there were significantly increased of the 5-HT concentration in plasma,while the DA concentration has a significant decreased?P<0.05?.At the same time,the 5-HT or DA receptors m RNA levels has a significantly changed in different tissues,including 5-HT1B,5-HT2B,5-HT7,DA1A and DA2.In addition,there was a significant change of CHH m RNA level in different tissues?P<0.05?and a significant decrease of glucose level in hemolymph.The above studies preliminarily showed that 5-HT and DA are involved in regulating the agonistic behavior through downstream receptors,and the occurrence of agonistic behavior is usually accompanied by energy consumption.At the same time,we selected the mating mode of male-male group as the follow-up study materials.In order to further study the molecular mechanism of agonistic behavior,we used the deep sequencing approach to identification and integrated analysis the micro RNA and m RNA expression profiles during agonistic behavior in E.sinensis.First,we divided the crabs into two groups:the control group?crabs without any fight experience?and the fight group?with fight experience?,then extracted the total RNA to construct the si RNA and m RNA libraries,and finally sequenced using Illumina Hiseq?2000.According to the results,we found 29 significant differentially expressed mi RNAs?DEMs?and 116 significant differentially expressed unigenes?DEGs?.The DEMs esi-mi R-199a-5p,esi-let-7d,esi-mi R-200a and esi-mi R-200b might participate in the regulation of agonistic behavior by mediating neuro-regulation and energy metabolism.Focusing on the transcripts of the m RNAs,the renin-angiotensin system KEGG pathway might be involved in the regulation of agonistic behavior through glucose metabolism as this pathway was significantly enriched with DEGs.Besides,an integrated analysis of the mi RNA and m RNA profiles revealed that the retinoid X receptor?RXR?was also involved in visual signal transduction,which was important for agonistic behavior.In addition,four vital agonistic behavior-related metabolic pathways,including the c AMP signaling,MAPK,protein digestion and absorption and fatty metabolism pathways,were significantly enriched with the predicted target unigenes.In conclusion,the findings of this study might provide important insight enhancing our understanding of the underlying molecular mechanisms of agonistic behavior in E.sinensis.According the above results,the 5-HT and DA levels has a significant changed before and after the fight,at the same time,the expression level of their downstream receptors also have a significant changed.In addition,the results of high-throughput sequencing showed that c AMP signaling pathway was significantly enriched predicted unigenes,and the c AMP,PKA in plasma also have a significant changed before and after the fight.The purpose of this study was to further explore the role of 5-HT and DA in regulating the agonistic behavior by activating c AMP signaling pathway through G-protein receptors using drug injection and in vitro culture.The results showed that injection of either 10-6 mol/crab 5-HT or DA reduced the agonistic behavior of E.sinensis?P<0.05?,as did 10-10 mol/crab DA,10-8 mol/crab 5-HT and DA?P<0.05?;however,a dose of 10-10 mol/crab 5-HT promoted agonistic behavior.5-HT significantly increased the m RNA expression level of 5-HT7 receptor and reduced that of the DA2 receptor in the cerebral ganglion?P<0.05?.In contrast to 5-HT,DA significantly decreased 5-HT2B m RNA levels and increased 5-HT7 and DA2 receptor levels in the thoracic ganglia?P<0.05?.In addition,injections of either 5-HT or DA increased the c AMP and PKA levels in hemolymph?P<0.05?.By using in vitro culture of the thoracic ganglia,the current study showed that ketanserin?5-HT2 antagonist?and[R?-?-TNPA]?DA2 agonist?had obvious effects on the expression levels of the two receptors?P<0.05?.In vivo experiments further demonstrated that ketanserin and[R?-?-TNPA]could both significantly reduce the agonistic behavior of the crabs?P<0.05?.However,ketanserin injection did not result in the significant decrease expression of 5-HT2B receptor,while[R?-?-TNPA]injection could significantly promote the expression of DA2 receptor.Furthermore,both ketanserin and[R?-?-TNPA]promoted the c AMP and PKA levels?P<0.05?.The injection of CPT-c AMP?c AMP analogue?elevated the PKA levels and inhibited agonistic behavior.In summary,this study showed that 5HT-2B and DA2 receptors were involved in the agonistic behavior that 5-HT/DA induced through the c AMP-PKA pathway in E.sinensis.In the above experiments,we found that 5-HT2 receptor inhibitor could decrease the agonistic behavior of crab,but could not inhibitor the expression of 5-HT2B.And5-HT2 receptor has two subtypes of 5-HT2A and 5-HT2B.In order to explore the function of 5-HT2B in agonistic behavior,first,we cloned and obtained the full-length sequence of 5-HT2B gene.The total length of 5-HT2B gene is 3127bp,with 236bp 5'-UTR?non coding region?,779bp 3'-UTR?non coding region?,and ORF?open reading frame?contains 2112bp?including termination codon?,encoding 703 amino acids.It was found that the amino acid sequence of 5-HT2B in E.sinensis was highly conserved with the Cancer borealis,Homarus americanus and Macrobrachium rosenbergii and so on.The phylogenetic tree analysis showed that 5-HT2B of E.sinensis and Cancer borealis were closely related.Combined with the mi RNA database,we found that mi R-143 could significantly inhibit the expression of 5-HT2B through in vitro culture,and further found that CO-transfection of mi R-143 and the 5-HT2B-WT vector could significantly inhibit the activity of luciferase.And then,It was found that injected mi R-143 could significantly inhibit 5-HT2B m RNA level after 48 hours,and further confirmed that mi R-143 could inhibit the 5-HT2B protein level after injection using western blotting.The results indicated mi R-143 could target the 5-HT2B 3'UTR.However,mi R-143 injection was failed to inhibit the agonistic behavior,indicated that inhibition of 5-HT2B expression could not inhibit the agonistic behavior in Chinese mitten crab. |
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