| Apolygus lucorum is one of the most common species of mirid bugs in China.It is a polyphagous herbivorous pest with more than 200 recorded host plants from 49 different families and causes great economic losses in different agricultural products.Olfaction plays important roles for this insect,since vital behaviors such as foraging,predation,mate recognition,and oviposition site selection are primarily dependent on the chemical communication and detection of blends of airborne molecules from the environment.Therefore,research on A.lucorum olfaction provides the opportunity to investigate the interactions between the insect and its environment.The first step toward understanding the molecular mechanism of olfactory perception process is to study olfaction-related genes such as odorant receptors(ORs),which encode the proteins that function in odorant molecular detection.Deorphanization of ORs involved in odor detection elevates our understanding about the olfactory system of this pest and may lead to develop a chemical ecology-based control strategy.The partial sequences of 11 putative odorant receptors,AluOR11,16,62,66,69,80,82,90,93,99,and 105 were first identified from an antennal transcriptome database which was constructed and annotated in the previous study by our laboratory.In this study,the full-length transcripts of selected ORs were further verified via the rapid amplification of cDNA ends(RACE)and then cloned.The relative expression levels of selected OR genes in six different tissues of male and female bugs were examined by qPCR.Xenopus leavis oocyte along with two electrode voltage clamp(TEVC)system was used to examine the functional characteristics of candidate ORs.Electroantennography(EAG)was used to record the antennal responses of 0-24 h old virgin male and female bugs to electro-physiologically active odorants from TEVC assay.The behavioral activity of compounds recorded from TEVC were evaluated by using the 3-cages olfactometer.RNA interference(RNAi)experiments were conducted to investigate the functions of selected AlucORs and AlucOrco in detecting different volatile.qPCR analysis and Y-tube olfactometer bioassay,respectively,were conducted to test the efficiency of RNAi experiments and possible effect of gene knocking down on A.lucorum adult's behavior.Bioinformatics analysis indicate that,similar to other insect species ORs,all candidate ORs contain seven putative transmembrane domains(TMDs)with an intracellular N-terminus and extracellular C-terminus except for AlucOR105 which has six putative transmembrane domains and an unusual structure(extracellular N-terminus and C-terminus).The multiple sequences alignment result indicated that ORs are extremely divergent in the same species and aligned sequences shared low amino acid identity.qPCR analysis revealed that all A.lucorum OR genes were enriched in the antennae compared to the other tissues.No significant differences were observed between males and females in the relative expression level of AlucOR11,AlucORI6,AlucOR82 and AlucOK93.For AlucOR62,AlucOR66 and AlucOR90,relative expression level in females were?1.5,2.5 and 1.5 fold higher than male,respectively.For AlucOR69,AlucOR80,AlucOR99 and AlucOR 105,male had significantly higher relative expression level(?2-5 fold)than female.Functional study demonstrated that except for AlucOR80,oocytes expressing other ORs did not elicit detectable currents when challenged with odorants in our test panel.AlucOR80/Orco-expressing oocytes successfully tuned to 21 components from different groups including esters,alcohols,ketones,monoterpenes and aldehydes.The EAG results indicated that all 21 candidate ligands could elicit electrophysiological responses in the male and female antennae.In behavioral trial,four out of 21 components including(1S)-(1)-beta-Pinene,l,8-Cineole,Dimethyl disulfide(DMDS)and S-(-)-cis-Verbenol showed repellent properties,among which DMDS was the most bioactive eomponent.In comparison,no behavioral responses were observed for 5 components including 1-pentadecanol,1-Tridecanol,Ethyl butyrate,Methyl anthranilate and Methyl phenylacetate.Other components were attractive for 4.lucorum adults among which,Butyl butyrate showed the highest attractiveness rate(65%for males and 72%for females).The RNAi assay analysis indicated that the reduction in the AlucOR80 transcript level increased with the increase of time and reached the highest level(?705 in males and?80%in females)72 and 96 h after dsOR80 injection.The behavioral responses of injected adults were not affected by either of the two repellents or two attractants in Y-tube assay and there was no significant differences between non-injected adults and dsOR80-injected adults.In a previous study,AlucOR28 responded to eight plant volatiles and shared 3 ligands with AlucOR80 including Butyl butyrate,Butyl propionate.As these two receptors share two important ligands which were shown to be behaviorally active for A.lucorum,we designed a double knock down RNAi to see the possible effects of silencing two receptors on the bug's behavior.As expected,the transcript levels of AlucOR28 and AlucOR80 significantly were reduced 72 and 96 h after double dsRNA injection.Surprisingly,the adults of this bug sowed preference to the volatile side in the Y-tube and no significant differences were observed between non-injected and double dsRNA-injected individuals.While RNAi knock down for AlucOK80 or double knock down for AlucOR28 and AlucOR80 failed to induce any behavioral effects on injected individuals,behavioral activities of A.lucorum was significantly affected in dsOrco treated insects,as compared with non-injected ones.On the other hand,the behavioral responses of adult bugs to Butyl butyrate and Dimethyl disulfide were sharply reduced due to reduction in the mRNA level of AlucOrco after dsOrco injectionAlthough our findings indicated that ten out of 11 tested AlucORs were silent in functional study,we cannot judge they are non-functional.Investigating their function with other methods might help to know more about these ORs.However,screening of AlucOR80 ligands by behavioral assay provided some basic information through which,olfactory-based management approaches could be developed by utilizing the behaviorally active components as attractants or repellents. |
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