Comparative Study Of Helicoverpa Armigera And Helicoverpa Assulta Counter-Defense Genes Lepidopteran Aminoacylase 1 And Glutathione S-Transferases

Helicoverpa armigera and Helicoverpa assulta are a pair of sibling species belonging to the Noctuidae family of Lepidoptra order.The two species represent two opposite evolutionary directions—generalization and specialization—in hostplant ranges although they are very similar in morphological and other biological characteristics.H.armigerais a highly polyphagous insect that can feed on over 200 plant speciesin more than 30 families,whereas H.assultais an oligophagous insect specializing on a few Solanaceae plants.To investigate why the two siblingspecies have such dramatically different host plant ranges,two types of counter-defense genes,the plant defense elicitor-hydrolyzing lepidopteran aminoacylase 1(L-ACY-1)and the plant defensive allelochemicals-detoxifying glutathione S-transferases(GSTs)were compared at both the genomic and transcriptomic levels between the two sibling species.The plant defense elicitors that L-ACY-1 hydrolyzes are fatty acid-amino acid conjugates(FACs)found in the oral secretions and frass of most lepidopteran larvae.Plants in Poaceae(maize),Fabaceae(soybean),Solanaceae(eggplant)and possibly additional families can recognize FACs and use them as signals to escalate their direct(e.g.anti-herbivore allelochemicals and proteinase inhibitors)and indirect defenses[natural enemies-attracting volatile organic compounds(VOCs)].Such a cost of triggering plant direct and indirect defenses can be offset by FACs’ benefit to insects—nitrogenassimilation by functioning not only as a sink for glutamine through depleting glutamine in midgut cells but also as a primary storage of glutaminesynthesized from glutamic acid and ammonia by glutamine synthetase.L-ACY-1 is the major gene that insect herbivores use to balance FACs’beneficial and detrimental effects.GSTs are one important group of allelochemicals-detoxifying enzymes in insect herbivores.The detrimental effects of allelochemicals to insects are variable,including repelling,antifeeding,production of reactive oxygen species(ROS,i.e.oxidative stress),disruption of growth and development and direct killing.GSTscan protect herbivores by directly metabolizing allelochemicals or detoxifying ROS derived from allelochemicals.Insect herbivores with more genes,alleles and/or isoforms of L-ACY-1 and GSTs of the two types of counter defense genes areexpected to possess greater counter defense capability.1.Genomic and transcriptional comparison of L-ACY-1 in the two sibling speciesTwo L-ACY-1 genes respectively present in the genomes of H.armigera and H.assulta were characterized and analyzed.Two L-ACY-1 genes shared the same deduced amino acid sequences,but there were two different amino acids between the two H.armigera L-ACY-1.Such divergences suggest thatH.Armigera L-ACY-1 couldhydrolyze more kinds of FACs which are composed of unsaturated fattyacid form plant and glutamine or glutamic acid from herbivores larva.At the same time,only L-ACY-1 transcriptwas respectively found in H.armigera and H.assulta transcriptomes.I failed to find the alleles or isforms due to the alternative splicing.In addition,intraspecific-interspecific comparison revealed that L-ACY-1 expression showed tissue specificity,the RPKM value was high in midgut and was very low in fatbody.Interspecific comparison revealed that the RPKM value was higher in the specialist H.assulta than the generalist H.armigera.2.Expressional divergence of L-ACY-1 in the two sibling speciesHow FAC-producing generalist and specialist herbivores regulate their FACs-hydrolyzing enzyme L-ACY-1 to balance FACs’ beneficial vs.detrimental effects remains unknown.To address this question,I compared L-ACY-1 expression in H.armigera and H.assul.ta by the same sets of hostplants and another two possible influence factors[protein to digestible carbohydrate(P:C)ratios or allelochemical which are really existed but different in hostplants].L-ACY-1 expression remained low/unchanged in H.armigera,but was induced by hot pepper fruits and repressed by cotton bolls in H.assulta.The representative allelochemicals of the tested hostplants significantly(capsaicin)or numerically(gossypol and nicotine)induced L-ACY-1 expression in H.armigera,but slightlyinhibited(capsaicin and gossypol)or induced(nicotine)it in H.assulta.L-ACY-1 expression remained low/unaltered on balanced(P50:C50 and P53:C47)or protein-biased diets and induced on carbohydrate-biased diets in H.armigera,but was at the highest level on balanced diets and reduced on either protein-or carbohydrate-biased diets in H.assulta.Furthermore,L-ACY-1 expression was significantly higher in H,assulta than in H.armigerafor most offeeding treatments.Such expressional divergences suggest that FACs are utilized mainly for removal of excessive nitrogen in generalists but for nitrogen assimilation in specialists.3.Genomic comparisonof GSTs gene in two sibling speciesThe genomes of H.armigera and H.assulta male pupae were fully sequenced and assembled.All the cytosolic GST genes present in the genomes of H.armigera and H.assulta were characterized andnamed according to their phylogenetic relationships and conventional nomenclature.Fifty functional GST genes and 2 GST pseudogene(52 in total)were identified in H.armigera,whereas 48 functional GST genes and 5 GST pseudogenes(53 in total)in H.assulta.Although the total numbers of GST genes were similar between the two species,the generalist H.armigera had 9 more Epsilon class GST genes but 3 fewer Delta class genes that are usually involved in detoxification of allelochemicals and insecticides,consistent with their host range difference.This indicates that more gene duplication events occurred in the Epsilon class but less in the Sigma and Delta class in H.armigera.The 5 Sigmaclass pseudogenes found in H.assulta and 2 Epsilon class pseudogenes in H.armigera were formed due to generation of pre-mature stop codon via mutation.In addition,I found 4 unique GSTs genes(HarmGSTS3、HarmGSTS4、HarmGSTS7 and HarmGSTS9 in H.armigera but no unique GSTs gene in H.assulta,which impliesthat the functions performed by the 4 H.armigera-unique GST genesare absent in the specialist H.assulta.4.Transcriptomic comparison of GSTs in the two sibling speciesRNA-seq was conducted to reveal the larval midgut and fatbody transcriptomes of the two species at the similar equencing depth.Overall,there were 42704 unigenes in H.armigera transcriptomes,but only 38241 unigenes in the H.assulta transcriptome,indicating that more genes were expressed in the generalist than in the specialist.This general trend held true for GSTs.A total of 69 GST unigenes were identified in the generalist transcriptomes,whereas only 47 GST unigenes were found in the specialist transcriptomes.Comparison of the unigenes with the genomic GST genes showed that 35 of the 50 H.armigera GST genes and 31 of the 48 H.assulta GST genes were expressed in the larval midguts and/or fatbody of the two species.The remaining 34 H.armigera GST unigenes and 14 H.assulta GST unigenes were the alleles or isoforms(derived from alternative transcription start site,alternative splicing,or alternative polyadenylation)of the above expressed GST genes and their fusion hybrids(derived from unequel crossing-over).Tweenty of the 34 H.armigera unigenes and 11 ofthe 14 H.assulta GST unigenes were the alleles of the expressed GST genes.Seven of the 34 H.armigera unigenes are fusion hybrids of the expressed GST genes,but there is no such kind of unigenes in H.assulata.The rest of unigenes were isoforms.I found 3 alternative splicing isoforms due to intron retain,exons skip,and alternative promoter/first exon in H.armigera,3 alternative splicing isoforms derived intron retain and 1 isoform from alternative promoter/first exon including in H.assulta.In addition,two H.armigera GST genes(HarmGSTE1 7 and HarmGSTE18)have mutually exclusive exons,but no H.assultaGST gene contains mutually exclusive exons.Taken together,finding of more expressed GST genes,more alleles,more fusion hybrids paralogs and more isoforms in H.armigera than H.assulta suggest that GST-based counter defense capacity is greater in the generalist H.armigera than the specialist H.assulta.