Molecular Mechanisms Underlying Color Preference Behavior Of Bactrocera Minax And Bactrocera Dorsalis Based On Omics Analysis

Fruit flies(Diptera:Tephritidae)are among the most economically important pests attacking fruits worldwide.Bactrocera minax is an oligophagous insect that oviposits only into the immature green fruits of Citrus species,where the larvae feed,resulting in huge economic losses.However,Bactrocera dorsalis is a polyphagous pest,with recorded host range of over 250 fruits and vegetables.Sexually mature females prefer the yellow mature hosts to lay eggs.Based on color preference behavior of tephritid species,various traps have been developed and used to control the pests,though with uneven effects.On the other hand,the molecular mechanisms underlying color preference behavior of insects are still unclear.As such,B.minax and B.dorsalis were selected as model insects to conduct the experiments in this study.Firstly,we generated de novo genome assemblies for the two tephtid fruit flies.Then semi-field cage behavioral assay was performed,we found that vision mediate color preference more importantly than olfaction in host location process of two Bactrocera species at close range.Furthermore,comparative genomic and transcriptome analyses was applied to identity opsin genes of two Bactrocera species.Base on RNAi technique and a series of behavioral experiment,we reveal the molecular mechanism of color preference behavior in B.minax and B.dorsalis.Our results provide basis for further understanding the molecular mechanism of host location and feeding habit of two tephritid species.The main results are as follows:1.Comparative genomic and population structure analysis of B.minax and B.dorsalisWe used Illumina sequencing platforms to generate data for assembling the genome assembly of B.minax.The assembled genome size of B.minax was 339 Mb,which comprised 1172 scaffolds with an N50 size of 9.8 Mb.Then Illumina and PacBio sequencing platforms were utilized for assembling the genome assembly of B.dorsalis.we found that the genome of B.dorsalis had an assembled size of 528 Mb,comprising 956 contigs with an N50 contig length of 1.2 Mb.Finilly,13878 and 16,741 genes were respectively annotated in B.minax and B.dorsalis.The completeness of coverage of our assembly was evaluated using software for quantitative measures for the assessment of two genomes assembly,which show 96.8%and 97.2%completeness in B.minax and B.dorsalis,respectively.We also found that the pathways of phototransduction and taste transduction experienced accelerated evolution in B.minax and B.dorsalis,respectively.Comparative genomic analyses inferred that B.minax may be evolved earlier.We further reconstructed the recent demographic history of B.minax and B.dorsalis using genome re-sequencing.In contrast to the steadily declining pattern in B.minax,the demography of B.dorsalis ended with a sudden expansion.In addition,we concluded that smoothend,FLASH,TfAP-2 and Lrrk were positive selection gene in B.minax,while Cyp6g2 was positive selection gene in B.dorsalis.Together,our results provide firstly the strong data for the study of gene function and molecular mechanism of color preference behavior of two Bactrocera species.2.Comparative transcriptome analysis of the heads of B.minax and B.dorsalisIn order to further explore the expression of vision related genes in different stages,we used Illumina HiSeq 4000 platform to perform transcriptome sequencing of two Bactrocera species.We obtain a total of 53.6 G high quality short sequences.Then transcriptome assembly was performed via two Bactrocera species genome.A total of 11615 and 14908 genes were annotated in B.minax and B.dorsalis,respectively.Finally,differentially expressed genes in newly emerged and sexually mature flies were determined based on both fold change.A total of 477 and 155 genes were found to be significantly upregulated in the sexually mature females of B.minax and B.dorsalis,respectively.Among them,all opsin genes in mature stage were significantly higher than newly emerged stage in two Bactrocera species.3.Comparison of the role of vision and olfaction in host location of B.minax and B.dorsalisTo compare the role of vision and olfaction in host location of two Bactrocera species at close range,we detected the spatio-temporal expression profile of Orco in B.minax and B.dorsalis.Our results show that Orco expression was significantly highter in sexually mature stage than newly emerged stage,and Orco expression was significantly highter in head than other tissues.In addition,based on RNAi,oviposition preference assay,electroantennogram test and Y-tube olfactometer assays analysis,we found that Orco involved in host location in B.minax and B.dorsalis.Furthermore,the expression of Orco was not affected by the silencing of opsin genes and vice versa.We designed a trap system in which the flies were given choices between single cues and combined cues,including the colour they prefer(B.minax:green,B.dorsalis:yellow),the colour they dislike(blue),and the odour they prefer(B.minax:the odor of unripe citrus juice,B.dorsalis:the odor of ripe citrus juice)but painted with the colour they dislike.In the wild cage experiments,both B.minax and B.dorsalis oriented significantly to the trap painted with their favourite color than the one in which their favourite odour was volatilized.We further performed tests in flies whose antennae had been surgically removed and those whose eyes had been painted with white acrylic paint.The antennaeless flies exhibited a reduced attraction rate but still strongly preferred to the trap with preferred color.However,a small proportion of eyeless flies could still locate a trap,without any significant locating preference.The above results suggest that vision may plays a more important role in the recognition and location of host in two Bactrocera species in short distance.4.Rh6 regulate the color preferences of B.minax and B.dorsalisFirsly,we screened opsin genes of B.minax and B.dorsalis by two Bactrocera species genome sequences.Rh1,Rh2,Rh3 and Rh6 were identified in two Bactrocera species.We found that the Rh5 opsin genes have been completely lost in B.minax and B.dorsalis.Moreover,Drosophilidae genomes encode two copies of ultraviolet-sensitive opsin genes Rh3 and Rh4 in Drosophila melanogaster,while in B.minax and B.dorsalis genomes only encode one Rh3 opsin gene at a single genomic locus comprisin two alternative splicing variants.Then the expression profile of each visual opsin gene throughout the adult stage were done by qPCR.Overall,Rh1,Rh2,Rh3 and Rh6 genes were found to be expressed at low levels in newly emerged adults and then experienced gradual elevation during development in both species.In addition,based on RNAi and system behavior experimental analysis,we found that BmRh6 regulate the preference for green in B.minax and BdRh6 regulate the preference for yellow in B.dorsalis.Multiple alignment analyses revealed that Rh6 was highly conserved between B.minax and B.dorsalis.Finally,we analyzed Hippo pathway,Phototransduction pathway and Eye development pathway gene copy number.A total of 12 genes presented different copy number.In conclusion,Illumina and PacBio sequencing platforms were utilized for assembling the genome assembly of two Bactrocera species.Our results shown that the genome of B.minax and B.dorsalis had the assembled size of 339 Mb and 528 Mb,respectivly.Then we also found that the pathways of phototransduction and taste transduction experienced accelerated evolution in B.minax and B.dorsalis,respectively.Rh1,Rh2,Rh3 and Rh6 were identified in two Bactrocera species.we found that the Rh5 have been completely lost in B.minax and B.dorsalis.Moreover,Rh3 and Rh4 only encode one UV opsin gene at a single genomic locus comprisin two alternative splicing variants.In addition,our results demonstrated that vision is more important than olfaction in host location process of two Bactrocera species at close range.Furthermore,we found that BmRh6 regulate the preference for green in B.minax and BdRh6 regulate the preference for yellow in B.dorsalis.Our results reveal the molecular mechanism of color preference behavior in B.mianx and B.dorsalis.The above results provide the theoretical basis for the molecular mechanism of insect host location and the scientific basis for the popularization and application of eco-friendly techniques for the control of the pest.