| The red swamp crayfish Procambarus clarkii is one globally aquatic-invasive species（AIS）,which caused massive destruction and threats to China’s agriculture,environment,and biodiversity.Currently,P.clarkii is widely distributed in China with rapid proliferation.Physical interruptions or chemical drug controls have a limited effect on its fast diffusion.Therefore,seeking an effective,environment-friendly,and targeted method to control P.clarkii has become the research emphasis.Sex pheromone control for pests is one of the mainstream technical means,which is applied mainly in agricultural-related pests and terrestrial invasive species,but rarely in AIS.For laying a foundation for follow-up prevention,it is urgent to strengthen the study on sex pheromones of AIS.In this study,we combined behavioral tests,EAG,and omics to screen the bioactive content of sex pheromones from female P.clarkii,and reveal the different mechanisms of sex pheromone reception and chemoreception between male and female P.clarkii,further providing the basic data for subsequent studies.The predominant contents of this study were as followed:（1）the identification of active pheromones from female P.clarkii;（2）the differences in chemoreception between male and female P.clarkii;（3）the differences in sensitivity of antennae for different stimuli between males and females.The results are as followed:（1）The semi preparative-HPLC technology was used to further isolate the bioactive fragments（interceptive samples）from female P.clarkii to 6 fragments（namely PPF1-PPF6）.The bioassay test results showed that only the PPF6 can significantly attract male P.clarkii and induce courtship behavior.（2）The preparative fragment 6 from male and female P.clarkii was further analyzed using UHPLC-MS/MS,for comparison of different metabolites（DMs）.The results of metabolic analysis had shown that 472 metabolites were identified from both male and female groups,of which 18 metabolites were filtrated as DMs（P<0.05）.Among these DMs,15 were significantly up-regulated and 3 were significantly down-regulated in female groups using the positive and negative ion models.（3）In the current study,6 endogenous and harmless DMs were selected from 18 DMs to perform the bioassay tests,including cis,cis-muconic acid,mestranol,2（1h）-pyridinone,creatinine,creatine,indole-3-acetonitrile.The results had shown that among 6 selected DMs,only the mestranol can significantly attract the male P.clarkii,but not females.In addition,the mestranol with different concentrations was used to determine the attraction to male P.clarkii.The effective attraction concentration of mestranol was 10-5-10-2 mol/L.The attraction increased with the concentration of mestranol.（4）Electroantennography（EAG）was used to determine the difference in stimulation potential of 8 stimuli（cis,cis-muconic acid,mestranol,2（1h）-pyridinone,creatinine,creatine,indole-3-acetonitrile,DMSO,and female conditioned water）on the antennae of P.clarkii.The EAG results had shown that all 8 stimuli had certain irritants to the antennae of P.clarkii,of which the female-conditioned water was the most predominant stimulus for males and cis,cis-muconic acid for females.Moreover,excluding the cis,cis-muconic acid,the electric potential values of male P.clarkii to the other 7 selected stimuli were all significantly higher than females.The paired samples t-test was used to analyze the intragroup electric potential values of male and female P.clarkii,which had shown that no significant difference in the electric potential values of DMSO-solubilized stimuli（DMSO,mestranol and indole-3-acetonitrile）in both male and female groups.（5）The antenna of P.clarkii was stimulated with mestranol at equal concentrations,and the transcriptome sequencing technology（RNA-Seq）was used to analyze the differently expression genes（DEGs）between male and female individuals.The DEGs analysis had shown that a total of 8192 DEGs had been identified between male and female groups（P<0.05）,of which 7939 DEGs were up-regulated and 253 down-regulated in male groups.The results of gene functional annotation had shown that a total of 5 types of chemosensory-related receptors and enzymes were found in both male and female groups,including ionotropic receptors（IRs）,olfactory receptors（ORs）,transient receptor potential cation channels（TRP channels）,gustatory receptors（GRs）,and pheromone processing carboxypeptidase,even though,the expressions of all these receptors and enzymes had no significant difference between male and female groups.The results of gene enrichment had shown that the DEGs were mainly enriched into signal transduction-and lipid metabolism-related pathways in the top 30 enriched pathways.In this study,we further analyzed the DEGs enriched in the c AMP signaling pathway,and which was found that 81 DEGs were enriched in total.Of these DEGs,80 DEGs were up-regulated in the c AMP signaling pathway（male groups）,while only one was down-regulated.Furthermore,a total of 16 DEGs were enriched to the calcium ion binding protein（CaM）,11 to calcium transport ATPase（PMCA）,and 10 to Sodium/Potassium pump（ATP）,indicating that a mass of DEGs in the cAMP signaling pathway was enriched to the proteins and enzymes that related to ion transport.This study has shown that mestranol was the main active component of sex pheromone in female P.clarkii.The difference in the chemosensitivity of male and female antennae stimulated by sex pheromones is mainly caused by ion transport activity,and lipids are involved in the pathway mediating ion signal transduction of P.clarkii antennae.At the same time,this study also provides reference ideas and a theoretical basis for the subsequent control of aquatic alien invasive organisms. |
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