Preliminary Studies On The Genomic Bases Of Polyphagy In Helicoverpa Armigera

The cotton bollworm,Helicoverpa armigera(Lepidoptera: Noctuidae),is a worldwide serious pest of agricultural importance.It is one of the most polyphagous insect,with a host range of more than 300 recorded plant species that belong to at least 60 families.Polyphagy is one of the most important factors contributing to its major pest status in agroecosystem.In recent years,the rapid development of DNA sequencing technology and bioinformatics has enabled genomics research methods to be widely applied to the field of entomology.Meanwhile,a large number of studies on molecular biology and transcriptomics of H.armigera have laid a good foundation for the launch of H.armigera genomics research.In this study,the genome sizes of H.armigera and its oligophagous sibling species,H.assulta were estimated by flow cytometry.Then,transcriptomes of larval midgut,fat body and adult antennae of H.armigera were sequenced by RNA-seq,and the obtained sequence reads were assembled with the publically available EST and RNA-seq reads to yield a merged H.armigera transcriptome.Third,Illumina and PacBio sequencing techniques were combined to sequence the genome of H.armigera.Finally,the gene families involved in host plant use were identified from the H.armigera genome and compared with their counterparts in the genomes of the oligophagous insects Bombyx mori and Manduca sexta to elucidate the phylogenetic relationships of the host plant use-related gene families and the molecular mechanism of polyphagy,which might provide theoretical reference for exploration of new pest management strategies.The main results of this dissertation can be summarized as follows:1.Flow cytometry revealed that both adult head of Drosophila melanogaster(the standard reference)and six larval tissues(head,midgut,hemolymph,salivary gland,male gonad and Malpighian tubule)of H.armigera not only had a cluster of diploid nuclei(2Cx),but also possessed at least two clusters of endopolyploid(4Cx and 8Cx)nuclei.Their nucleus composition displayed three different patterns: decreased with the round of endoreplication(larval head,salivary gland,and Malpighian tubule of H.armigera and adult head of D.melanogaster),increased from 2Cx to the highest endopolypolid level(larval hemolymph of H.armigera),or increased with the round of endoreplication to the second highest endopolypolid level and then dropped sharply at the highest endopolypolid level(larval male gonad and midgut of H.armigera).The C-values estimated with six larval tissues of H.armigera varied significantly,indicating the importance of materials used for genome size estimate.Thus,larval heads were selected for the subsequent determination in consideration of result errors and tissue consistency.The genome size of H.armigera is 394 Mb,which is larger than that of H.zea(363Mb),a generalist derived from H.armigera,but smaller than that of H.assulta(430 Mb),the common oligophagous ancestor of the two polyphagous species H.armigera and H.zea.The results indicate that genome size tends to contract but host plant range is inclined to expand in the terminal Helicoverpa lineage.2.RNA-seq was conducted to obtained the transcriptomes of the tissues/organs involved in selection(female and male antennae of H.armigera adults)or utilization(larval midgut,fat body of H.armigera)of host plant.The resultant reads were then merged with the RNA-seq reads of 17 transcriptomes and 30,532 EST sequences of H.armigera downloaded from the NCBI database as of October 2015,and reassembled into a fusion transcriptome of 178.4 Mb,containing 129,027 transcripts with an N50 of 3016 bp.Further cluster analysis indicates that the 129,027 transcripts represent 53,881 unigenes.Comparative analysis of the core statistic parameters shows that the fusion transcriptome is significantly better in terms of information volume,assembly quality and completeness than all the individual transcriptome.3.The genome of H.armigera were completely sequenced by combination of the second-and third-generation DNA sequencing techniques.The size of the assembled H.armigera genome was 402 Mb,consisting of 1776 scaffolds with an N50 of 497.2 kb.The GC content in the genome was 36.5%.The proportion of repeat sequences was 22.2%.The genome was predicted to encode a total of 20398 protein-coding genes through genome annotation.The quality assessment results showed that 92% of transcripts in the H.armigera fusion transcriptome and 92% of the BUSCO arthropod single-copy genes were covered by the assembled H.armigera genome,indicating that the genome was well assembled.4.Gene families and their members involved in chemoreception and detoxification metabolism were identified in the genome of H.armigera by manual annotation.Among the identified chemoreception genes were 44 odorant-binding proteins(OBPs),35 chemosensory proteins(CSPs),80 odorant receptors(ORs),33 ionotropic receptors(IRs),4 sensory neuron membrane proteins(SNMPs),and 185 gustatory receptors(GRs).The identified detoxification genes included 112 cytochrome P450 s,92 esterases,50 glutathione S-transferases(GSTs)and 64 ATP-binding cassette transporters(ABC transporters).Compared with the chemoreception and detoxification genes in the genomes of the oligophagous B.mori and M.sexta,bitter receptors of GRs,Clan 3 and Clan 4 P450 s,Group E esterases,Epsilon GSTs,ABCC and ABCG transporters were significantly expanded / duplicated in the genome of the polyphagous H.armigera.The expansions of the aforementioned chemoreception and detoxification genes should contribute,at least partially,to the capacity of H.armigera to feed on hundreds of distinct host plants and the evolution of its survival strategy of polyphagy.