Molecular Mechanisms Of Neuroendocrine Regulatory Effect On Locomotor And Stress Behavior In Apostichopus Japonicus

Apostichopus japonicus is an important commercial marine species in China.The single production value of A.japonicus aquaculture industry is the largest one in all the marine aquaculture industries in northern China.The behavioral research of sea cucumber can provide reference for the development of sea cucumber catching facilities and sea cucumber enhancement and releasing strategies,and provide data support for the innovation of sea cucumber aquaculture pattern including industrial and pond aquaculture.Behavioral endocrinology is an important branch of behavioral research that mainly study the interaction between hormones and behavior in animals.In this study,melatonin and two representative neuropeptides were used to study their effects on locomotor behavior of A.japonicus by infrared camera and ethovision software.Meanwhile,metabolomics was used to systematically detect the changing of muscle physiology after melatonin and two neuropeptides administration.In part,we expected to clarify the intrinsic physiological mechanisms of the behavioral regulation of melatonin and two neuropeptides.In addition,transcriptomics techniques were used to identify the intrinsic molecular mechanisms of sea cucumber evisceration behavior.1.The effect of melatonin on locomotor behavior and muscle physiology in A.japonicusThe goals of this part were to show the existence of melatonin in the sea cucumber A.japonicus and to evaluate its effect on locomotor activity.In addition,muscle tissues from control and melatonin-treated sea cucumbers were tested using ultra performance liquid chromatography and quadrupole time-offlight mass spectrometry(UPLC-QTOF-MS)to determine the changes of metabolic activity in muscle.Melatonin was present in the coelomic fluid of A.japonicus at a concentration of ～135.0 ng/L.The total distance traveled and number steps taken over 9 h after melatonin administration decreased with increasing concentration of the melatonin dose.Mean and maximum velocity of movement and stride length and stride frequency also decreased,but their differences were not statistically significant.Overall,these results suggest that melatonin administration had a sedative effect on A.japonicus.The levels of 22 different metabolites were altered in the muscle tissues of melatonin-treated sea cucumbers.Serotonin,9-cis retinoic acid,all-trans retinoic acid,flavin mononucleotide in muscles were downregulated after melatonin administration.Moreover,a high free fatty acid(FFA)concentration and a decrease in the adenosine 50-triphosphate(ATP)concentration in the muscle tissues of the melatonin-treated group were detected as well.These results suggest that the sedative effect of melatonin involves some other metabolic pathways,and the reduced locomotor modulator— serotonin,inhibited fatty acid oxidation and disturbed oxidative phosphorylation are potential physiological mechanisms that result in the inhibitory effect of melatonin on locomotion in sea cucumbers.2.The effect of pedal neuropeptide on locomotor behavior and muscle physiology in A.japonicusThe changing of A.japonicus locomotor behavior was recorded after pedal neuropeptide administration in this part.In addition,UPL-Q-TOF-MS metabolomics was used to detect the changing of muscle metabolites after pedal neuropeptide injection.The results showed that the locomotor stride of A.japonicus after pedal neuropeptide injection was decreased,indicating that this neuropeptide was likely to participate in the regulation of muscle contraction in A.japonicus.Besides,the increase of moving distance and steps and the decrease of moving velocity indicated that pedal neuropeptide can enhance the moving tolerance and reduce the moving efficiency in sea cucumber.The data of muscle metabolomics suggested that down-regulation of phosphatidylethanolamine(PE)and phosphatidylcholine(PC),up-regulation of LysoPCs,LysoPEs and arachidonic acid(ARA)might be the underlying mechanisms that responsible for behavioral effects of pedal neuropeptide in A.japonicus.3.The effect of L-SALMFamide neuropeptide on locomotor behavior and muscle physiology in A.japonicusAfter synthesizing L-type SALMFamide neuropeptide in vitro,the regulation of SALMFamide neuropeptide on locomotor behavior of A.japonicus was studied by neuropeptide coelom injection.Besides,the changes of muscle physiology were detected by metabolomics.Our results showed that SALMFamide neuropeptide can increase the moving stride of sea cucumber,which indicates that the neuropeptide may take part in the muscle relaxation of A.japonicus.In addition,the increase of moving distance,number of steps,cumulative duration of moving and moving velocity indicated that SALMFamide neuropeptide enhanced not only the moving endurance,but also the moving efficiency of A.japonicus.Metabolomics results suggested that the increase of pantothenic acid,LysoPEs and arachidonic acid(ARA)concentration,as well as the changing of PC : PE ratio,were the potential physiological mechanisms underlying the regulation of this neuropeptides on locomotor behavior in A.japonicus.4.Molecular mechanisms responsible for evisceration behavior in A.japonicusIn this part,Illumina sequencing(RNA-Seq)was performed on A.japonicus specimens in three states: normal(TCQ),eviscerating(TCZ),and 3 h after evisceration(TCH).In total,129,905 unigenes were generated with an N50 length of 2651 base pairs,and 54,787 unigenes were annotated from seven functional databases(KEGG,KOG,GO,NR,NT,Interpro,and Swiss-Prot).Additionally,190,191,and 320 genes were identifed as differentially expressed genes(DEGs)in the comparisons of TCQ vs.TCZ,TCZ vs.TCH,and TCQ vs.TCH,respectively.These DEGs mapped to 157,113,and 190 signaling pathways in the KEGG database,respectively.KEGG analyses also revealed that potential DEGs enriched in the categories of “environmental information processing,” “organismal system,” “metabolism,” and “cellular processes,” and they were involved in evisceration behavior in A.japonicus.These DEGs are related to muscle contraction,hormone and neurotransmitter secretion,nerve and muscle damage,energy support,cellular stress,and apoptosis.In conclusion,through our comparative analysis of A.japonicus in different stages,we identifed many candidate eviscerationrelated genes and signaling pathways that likely are involved in evisceration behavior.These results should help further elucidate the mechanisms underlying evisceration behavior in sea cucumbers.