Dynamics and diversity of symbiotic bacteria in Apolygus lucorum at different developmental stages

Background Apolygus lucorum is a worldwide omnivorous pest damaging a range of crops and causing great economic losses.Symbiotic bacteria living in insects play a key role in the nutrition,physiology,and behavior of hosts.Here,we present an experiment using Illumina HiSeq sequencing targeting the V3–V4 regions of bacteria’s 16S rRNA throughout the entire life cycle of A.lucorum.Results The first and second instar nymphs have the largest alpha diversity compared with other life stages of the insect.Bacterial phyla Proteobacteria(72.29%),Firmicutes(15.24%),Actinobacteria(7.76%)exhibit the largest relative abundance in all developmental stages.Erwinia(23.97%)and Lactococcus(10.62%)are the two genera with the high-est relative abundance.The relative abundance of Erwinia in the nymph stage is significantly greater than the adult stage,and the relative abundance of Lactococcus in 6-day-old and 9-day-old adult females is higher compared with adult males.Conclusions These results reveal that microbial community composition and relative abundance shift dynamically at different life stages,implying that different bacterial phyla and genera may have specific roles in specific life stages such as metabolism,nutrition absorption,detoxification,and reproduction.This study reveals for the first time the community composition and ecological dynamics of symbiotic bacteria throughout the life stages of A.lucorum,and thus may provide insight to new strategies for pest control.

Control Efficacy of Botanical Pesticides Against Apolygus lucorum(Meyer-Dür.) and Erythroneura apicalis(Nawa) for Grape

Apolygus lucorum（Meyer-Dür.） and Erythroneura apicalis（Nawa） are important pests that affect the quality and the yield of grapevine and cause huge economic losses. This paper focuses on the selection of effective botanical pesticides to control A. lucorum and E. apicalis. This experiment explores the effect of several botanical pesticides for A. lucorum and E. apicalis, including the 0.5% veratrine, the0.6% Oxygen·Lactone agent, the 5% natural pyrethrin, the composite neem pesticide, the rotenone and the composite nicotine. The 0.5% veratrine shows a stable control efficacy, which is higher than 60% in Chengdu, while the composite nicotine shows the highest efficacy against A. lucorum, which is above 70%. In Yinchuan,the 0.5% veratrine shows the highest efficacy, against the first generation adults and the second generation larvae of E. apicalis, while the 5% natural pyrethrin shows 100% control efficacy against E. apicalis in Nanjiang. The 0.5% veratrine and the composite neem could be used as effective pesticides to control A. lucorum and the 5% natural pyrethrin can be used to control E. apicalis. They could be widely used in the production of pollution-free grapes.

Biosynthesis of artemisinic acid in engineered Saccharomyces cerevisiae and its attractiveness to the mirid bug Apolygus lucorum

Artemisia annua is an important preferred host of the mirid bug Apolygus lucorum in autumn.Volatiles emitted from A.annua attract A.Iucorum.Volatile artemisinic acid of A.annua is a precursor of artemisinin that has been widely investigated in the Chinese herbal medicine field.However,little is known at this point about the biological roles of artemisinic acid in regulating the behavioral trends of A.lucorum.In this study,we collected volatiles from A.annua at the seedling stage by using headspace solid phase microextraction(HS-SPME).Gas chromatography-mass spectrometry(GC-MS) analysis showed that approximately 11.03±6.00 and 238.25±121.67 ng hartemisinic acid were detected in volatile samples and milled samples,respectively.Subsequently,a key gene for artemisinic acid synthesis,the cytochrome P450 gene cyp71 av1,was expressed in engineered Saccharomyces cerevisiae to catalyze the production of artemisinic acid.After the addition of exogenous artemisinic alcohol or artemisinic aldehyde,artemisinic acid was identified as the product of the expressed gene.In electroantennogram(EAG) recordings,3-day-old adult A.lucorum showed significant electrophysiological responses to artemisinic alcohol,artemisinic aldehyde and artemisinic acid.Furthermore,3-day-old female bugs were significantly attracted by artemisinic acid and artemisinic alcohol at a concentration of 10 mmol L,whereas 3-day-old male bugs were attracted significantly by 10 mmol Lartemisinic acid and artemisinic aldehyde.We propose that artemisinic acid and its precursors could be used as potential attractant components for the design of novel integrated pest management strategies to control A.lucorum.

Suppression Subtractive Hybridization Reveals Different Responses of Two Varieties of Gossypium arboreum L. Under Apolygus lucorum Stress

Plants reshape their transcriptomes, proteomes and metabolomes in response to insect damage. In this study, we used suppression subtractive hybridization to investigate the transcriptomes of two cotton varieties （CCRI41 and CCRI23） under Apolygus lucorum damage. From the CCRI23 libraries we obtained 92 transcripts and from the CCRI41 libraries we obtained 96 transcripts. 26 and 63 of the transcripts from CCRI23 and CCRI41, respectively, had known functions. Using reverse transcription PCR, we detected expression proifle of genes with known functions. Ultimately, we identiifed eight signiifcantly regulated genes, including one downregulated and four upregulated genes from the CCRI41 libraries, and one downregulated and two upregulated genes from the CCRI23 libraries. Only the gene encoding the polyphenol oxidase （PPO） is involved in plant defense against insect herbivores, and the others are related to improving tolerance to insect damage. Quantitative real-time PCR was used to study changes in expression levels during A. lucorum damage in CCRI23 and CCRI41. Signiifcantly regulated genes from CCRI23 showed a response in CCRI23 but not response in CCRI41. Similarly, signiifcantly regulated genes from CCRI41 showed a response in CCRI41 but not response in CCRI23. The results showed that, among transcriptomes of cotton varieties, there are different responses to A. lucorum damage.

Occurrence and Pollution-free Control of Apolygus lucorum in Winter Jujube

In recent years, Apolygus htcorum has become the main pest of winter jujube. It sucks juice of young parts of winter jujube and causes falling of buds and flowers, leaf perforations, deformities and fall- off of fruits, leading to serious economic loss. Considering the occurrence characteristics and control problems, the distribution, occurrence regularity, damage characteristics and outbreak reasons of A. lucorum are overviewed and analyzed in this paper, and the pollution-free control measures are also put forward.

Gene cloning,protein expression,and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum

RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic cells.However,silencing efficacy varies greatly among different insect species.Recently,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection.The disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi efficiency.Here,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of A.lucorum(AldsRNase)was identified and characterized.Sequence alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’dsRNases.The signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali dsRNase.AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole body.The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA.When comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is dsRNA.Subsequently,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells.Through cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in A.lucorum and related species.

A de novo transcriptomic analysis to reveal functional genes in Apolygus lucorum

The widespread planting of genetically engineered cotton producing the CrylAc toxin has led to significantly reduced pesticide applications since 1997. However, conse- quently, the number of green mirid bugs （GMB）, Apolygus lucorum （Meyer-Diir） has in- creased. So far the GMB, instead of the cotton bollworm Helicoverpa armigera （Hiibner）, has become the major pest in the transgenic Bt cotton field and has influenced cotton yield. Disproportionately, only a few studies on GMB at molecular level have been re- ported. Libraries from both third instar nymphs and adults were sequenced using Illumina technology, producing more than 106 million short reads and assembled into 63 029 uni- genes of mean length 597 nt and N50 813 nt, ranging from 300 nt to 9771 nt. BLASTx analysis against Nr, Swissprot, GO and COG was performed to annotate these unigenes. As a result, 26 478 unigenes （42.01%） matched to known proteins and 107 immune-related, 320 digestive-related and 53 metamorphosis-related genes were detected in these annotated unigenes. Additionally, we profiled gene expression using mapping based differentially expressed genes （DEGs） strategy between the two developmental stages： nymph and adult. The results demonstrated that thousands of genes were significantly differentially ex- pressed at different developmental stages. The transcriptome and gene expression data provided comprehensive and global gene resources of GMB. This transcriptome would improve our understanding of the molecular mechanisms of various underlying biological characteristics, including development, digestion and immunity in GMB. Therefore, these findings could help elucidate the intrinsic factors of the GMB resurgence, offering novel pest management targets for future transgenic cotton breeding.

Transcriptome analysis of three cotton pests reveals features of gene expressions in the mesophyll feeder Apolygus lucorum

The green mirid bug Apolygus lucorum is an agricultural pest that is known to cause damage to more than 150 plant species. Here, we report the transcriptomes ofA. lucorum at three different developmental stages （the second and fifth instar nymphs and adults）. A total of 98,236 unigenes with an average length of 1,335nt was obtained, of which 50,640 were annotated, including those encoding digestive enzymes and cytochrome P450s. Comparisons with cotton bollworm and cotton aphid transcriptomes revealed distinct features ofA. lucorum as a mesophyll feeder. The gene expression dynamics varied during development from young nymphs to adults. The high-quality transcriptome data and the gene expression dynamics reported here provide valuable data for a more comprehensive understanding of the physiology and development of mirid bugs, and for mining targets for their control.

The mitochondrial genome of the plant bug Apolygus lucorum （Hemiptera： Miridae）＂ Presently known as the smallest in Heteroptera

The complete mitochondrial （mt） genome of the plant bug, Apolygus lucorum, an important cotton pest, has been sequenced and annotated in this study. The entire circular genome is 14 768 bp in size and represents the smallest in presently known heteropteran mt genomes. The mt genome is encoding for two ribosomal RNA （rRNA） genes, 22 transfer RNA （tRNA） genes, 13 protein coding genes and a control region, and the order, content, codon usage and base organization show similarity to a great extent to the hypothetical ancestral model. All protein coding genes use standard initiation codons ATN. Conventional stop codons TAA and TAG have been assigned to the most protein coding genes; however, COIII, ND4 and ND5 genes show incomplete terminator signal （T）. All tRNA genes possess the typical clover leaf structure, but the dihydrouridine arm of tRNAser（A6N） only forms a simple loop. Secondary structure models of rRNA genes are generally in accordance with the former models, although some differences exist in certain parts. Three intergenic spacers have never been found in sequenced mt genomes of Heteroptera. The phylogenetic study based on protein coding genes is largely congruent with previous phylogenetic work. Both Bayesian inference and maximum likelihood analyses highly support the sister relationship ofA. lucorum and Lygus lineolaris, and Miridae presents a sister position to Anthocoridae.

Identification of heat shock cognate protein 70 gene （Alhsc70） of Apolygus lucorum and its expression in response to different temperature and pesticide stresses

Heat shock cognate protein 70 （Hsc70） is a very important stress-resistance protein of insects against environmental stresses. We employed fluorescent real-time quan- titative polymerase chain reaction and Western-blot techniques to analyze the transcrip- tional and translational expression profiles of AIHSC70 under extreme temperature （4℃ and 40℃） or 4 pesticide stresses in Apolygus lucorum. The results showed that the expres- sion of AIHSC70 were significantly induced by cyhalothrin or extremely high temperature （40℃） in both transcriptional and translational levels （P 〈 0.05）, while the transcriptional and translational level of AIHSC70 decreased significantly in treatments of chlorpyrifos or extreme cold temperature （4℃） （P 〈 0.05）. Moreover, afterApolygus lucorum treated by imidacloprid or emamectin benzoate, the expression of AIHSC70 was only up-regulated significantly at the transcriptional level （P 〈 0.05）, although obviously up-regulated at the translational level of AIHSC70. Therefore, this study confirmed that the Alhsc70 gene played important roles in response to both temperature and pesticide stresses, especially for cyhalothrin or extremely high temperature （40℃）. In addition, the significant polynomial regression correlations between temperature and the Alhsc70 expression level were shown in all the nymph and adult stages （P 〈 0.01）, indicating temperature was an important factor to affect the relative expression of Alhsc70.

不同处理对绿盲蝽与小贯小绿叶蝉诱捕效果对比

运用不同色板、诱捕器对小贯小绿叶蝉(Empoasca onukii Matsuda)和绿盲蝽(Apolygus lucorum)进行诱捕。结果表明,用天敌友好型诱虫色板和黄板防治小贯小绿叶蝉、绿板防治绿盲蝽效果较好,初步掌握了襄阳市2种刺吸类害虫的发生情况。

冀南地区酸枣绿盲蝽种群动态监测

为有效控制绿盲蝽(Apolygus lucorum)对酸枣(Ziziphus jujuba var.spinosa)的危害,2019年对邢台地区酸枣绿盲蝽种群动态进行了监测,发现该害虫在酸枣树上的发生高峰出现在6月中旬和9月下旬,在枣园周边杂草上的发生高峰在7月下旬和9月中下旬。酸枣绿盲蝽可能也具有在酸枣树与周边杂草2种寄主间相互转移的习性。

Mirids secrete a TOPLESS targeting protein to enhance JA-mediated defense and gossypol accumulation for antagonizing cotton bollworms on cotton plants

Most coexisting insect species exhibit stunted growth compared to individual species on plants.This phenomenon reflects an interspecific antagonism drawing extensive attention,while the underlying mechanisms remain largely uncharacterized.Mirids(Apolygus lucorum)and cotton bollworms(Helicoverpa armigera)are two common cotton pests.We identified a secretory protein,ASP1,from the oral secretion of mirids,found in the nucleus of mirid-infested cotton leaves.ASP1 specifically targets the transcriptional co-repressor TOPLESS(TPL)and inhibits NINJA-mediated recruitment of TPL,promoting plant defense response and gossypol accumulation in cotton glands.ASP1-enhanced defense inhibits the growth of cotton bollworms on cotton plants,while having limited impact on mirids.The mesophyll-feeding characteristic allows mirids to avoid most cotton glands,invalidating cotton defense.Our investigation reveals the molecular mechanism by which mirids employ cotton defense to selectively inhibit the feeding of cotton bollworms.