Cloning and reproductive regulation of a trypsin precursor gene in Adelphocorissuturalis.

Adelphocoris suturalis is a major pest of cotton. Here, we identified a trypsin precursor gene (AsTryP) in A. suturali, which has an open reading frame region of 873 bp and belongs to the trypsin superfamily. The mRNA of the AsTryP gene was detectable in every life stage and different tissues of 8-day-old females, and the gene was highly expressed in fourth-instar nymphs and the thorax of 8-day-old females. Down-regulation of AsTryP by the injection of double-stranded RNA suppressed the ovarian development and female fertility. These results reveal that trypsin precursor is involved in the reproductive process of A. suturali, and may facilitate the development of new strategies for a better control of A. suturalis.

Expression Profiles of Digestive Genes in the Gut and Salivary Glands of Tarnished Plant Bug (Hemiptera: Miridae).

Host plant preference of agricultural pests may shift throughout the growing season, allowing the pests to persist on wild hosts when crops are not available. Lygus Hahn (Hemiptera: Miridae) bugs are severe pests of cotton during flowering and fruiting stages, but can persist on alternative crops, or on weed species. Diversity of digestive enzymes produced by salivary glands and gut tissues play a pivotal role in an organism's ability to utilize various food sources. Polyphagous insects produce an array of enzymes that can process carbohydrates, lipids, and proteins. In this study, the digestive enzyme repertoire of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by high-throughput sequencing followed by cDNA cloning and sequencing. This study identified 87 digestive genes, including 30 polygalacturonases (PG), one ß-galactosidase, three α-glucosidases, six ß-glucosidases, 28 trypsin-like proteases, three serine proteases, one apyrase-like protease, one cysteine protease, 12 lipases, and two transcripts with low similarity to a xylanase A-like genes. RNA-Seq expression profiles of these digestive genes in adult tarnished plant bugs revealed that 57 and 12 genes were differentially expressed in the salivary gland and gut (≥5-fold, P ≤ 0.01), respectively. All polygalacturonase genes, most proteases, and two xylanase-like genes were differentially expressed in salivary glands, while most of the carbohydrate and lipid processing enzymes were differentially expressed in the gut. Seven of the proteases (KF208689, KF208697, KF208698, KF208699, KF208700, KF208701, and KF208702) were not detected in either the gut or salivary glands.

Dual oxidase enables insect gut symbiosis by mediating respiratory network formation.

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.

Genetic diversity analysis of brown marmorated stink bug, Halyomorpha halys based on mitochondrial COI and COII haplotypes.

BACKGROUND: In the past decade, the brown marmorated stink bug (BMSB), Halyomorpha halys (Hemiptera: Pentatomidae) has caused extensive damage to global agriculture. As a high-risk pest for many countries, including New Zealand, it is important to explore its genetic diversity to enhance our knowledge and devise management strategies for BMSB populations. In this study, two mitochondrial genes, Cytochrome c oxidase I (COI) and Cytochrome c oxidase II (COII) were used to explore the genetic diversity among 463 BMSB individuals collected from 12 countries. RESULT: In total, 51 COI and 29 COII haplotypes of BMSB were found, which formed 59 combined haplotypes (5 reported and 54 novel). Of these, H1h1 was the predominant haplotype. The haplotype diversity (Hd) and nucleotide diversity (π) were high while the neutrality (Fu's Fs) values were negative for the BMSB populations in the native countries, China, and Japan. For the BMSB populations from the invaded countries, the Fu's Fs values were negative for populations from Chile, Georgia, Hungary, Italy, Romania, Turkey, and USA, indicating that those populations are under demographic expansion. In comparison, the Fu's Fs values were positive for the populations from Austria, Serbia, and Slovenia, revealing a potential population bottleneck. Analysis of molecular variance (AMOVA) suggested that significant genetic difference exists among the BMSB populations from China, Japan, and the invasive countries. CONCLUSION: This study revealed that the haplotype diversity of the BMSB populations was high in those two studied countries where BMSB is native to (China and Japan) but low in those countries which have been invaded by the species. The analysis indicated that multiple invasions of BMSB occurred in Europe and the USA. The study also revealed three ancestral lines and most of the novel haplotypes were evolved from them. Moreover, we observed two genetic clusters in the invasive populations that are formed during different invasion events. Our study provided a comprehensive overview on the global haplotypes distribution thus expanding the existing knowledge on BMSB genetic diversity that potentially could play an important role in formulating feasible pest management strategies.

Cathepsins L and B in Dysdercus peruvianus, Rhodnius prolixus, and Mahanarva fimbriolata. Looking for enzyme adaptations to digestion.

Cysteine peptidases (CP) play a role as digestive enzymes in hemipterans similar to serine peptidases in most other insects. There are two major CPs: cathepsin L (CAL), which is an endopeptidase and cathepsin B (CAB) that is both an exopeptidase and a minor endopeptidase. There are thirteen putative CALs in Dysdercus peruvianus, which in some cases were confirmed by cloning their encoding genes. RNA-seq data showed that DpCAL5 is mainly expressed in the anterior midgut (AM), DpCAL10 in carcass (whole body less midgut), suggesting it is a lysosomal enzyme, and the other DpCALs are expressed in middle (MM) and posterior (PM) midgut. The expression data were confirmed by qPCR and enzyme secretion to midgut lumen by a proteomic approach. Two CAL activities were isolated by chromatography from midgut samples with similar kinetic properties toward small substrates. Docking analysis of a long peptide with several DpCALs modeled with digestive Tenebrio molitor CAL (TmCAL3) as template showed that on adapting to luminal digestion DpCALs (chiefly DpCAL5) changed in relation to their ancestral lysosomal enzyme (DpCAL10) mainly at its S2 subsite. A similar conclusion arrived from structure alignment-based clustering of DpCALs based on structural similarity of the modeled structures. Changes mostly on S2 subsite could mean the enzymes turn out less peptide-bond selective, as described in TmCALs. R. prolixus CALs changed on adapting to luminal digestion, although less than DpCALs. Both D. peruvianus and R. prolixus have two digestive CABs which are expressed in the same extension as CALs, in the first digestive section of the midgut, but less than in the other midgut sections. Mahanarva fimbriolata does not seem to have digestive CALs and their digestive CABs are mainly expressed in the first digestive section of the midgut and do not diverge much from their lysosomal counterparts. The data suggest that CABs are necessary at the initial stage of digestion in CP-dependent Hemipterans, which action is completed by CALs with low peptide-bond selectivity in Heteroptera species. In M. fimbriolata protein digestion is supposed to be associated with the inactivation of sap noxious proteins, making CAB sufficient as digestive CP. Hemipteran genomes and transcriptome data showed that CALs have been recruited as digestive enzymes only in heteropterans, whereas digestive CABs occur in all hemipterans.

A transcriptomic and proteomic atlas of expression in the Nezara viridula (Heteroptera: Pentatomidae) midgut suggests the compartmentalization of xenobiotic metabolism and nutrient digestion.

BACKGROUND: Stink bugs are an emerging threat to crop security in many parts of the globe, but there are few genetic resources available to study their physiology at a molecular level. This is especially true for tissues such as the midgut, which forms the barrier between ingested material and the inside of the body. RESULTS: Here, we focus on the midgut of the southern green stink bug Nezara viridula and use both transcriptomic and proteomic approaches to create an atlas of expression along the four compartments of the anterior-posterior axis. Estimates of the transcriptome completeness were high, which led us to compare our predicted gene set to other related stink bugs and Hemiptera, finding a high number of species-specific genes in N. viridula. To understand midgut function, gene ontology and gene family enrichment analyses were performed for the most highly expressed and specific genes in each midgut compartment. These data suggested a role for the anterior midgut (regions M1-M3) in digestion and xenobiotic metabolism, while the most posterior compartment (M4) was enriched in transmembrane proteins. A more detailed characterization of these findings was undertaken by identifying individual members of the cytochrome P450 superfamily and nutrient transporters thought to absorb amino acids or sugars. CONCLUSIONS: These findings represent an initial step to understand the compartmentalization and physiology of the N. viridula midgut at a genetic level. Future studies will be able to build on this work and explore the molecular physiology of the stink bug midgut.

Diversity and function of multicopper oxidase genes in the stinkbug Plautia stali.

Multicopper oxidase (MCO) genes comprise multigene families in bacteria, fungi, plants and animals. Two families of MCO genes, MCO1 (laccase1) and MCO2 (laccase2), are conserved among diverse insects and relatively well-characterized, whereas additional MCO genes, whose biological functions have been poorly understood, are also found in some insects. Previous studies reported that MCO1 participates in gut immunity and MCO2 plays important roles in cuticle sclerotization and pigmentation of insects. In mosquitoes, MCO2 was reported to be involved in eggshell sclerotization and pigmentation, on the ground that knockdown of MCO2 caused deformity and fragility of the eggshell. Here we identified a total of 7 MCO genes, including PsMCO1 and PsMCO2, and investigated their expression and function in the brown-winged green stinkbug Plautia stali. RNA interference (RNAi) knockdown of MCO genes by injecting double-stranded RNA (dsRNA) into nymphs revealed that MCO2, but not the other 6 MCOs, is required for cuticle sclerotization and pigmentation, and also for survival of P. stali. Trans-generational knockdown of MCO2 by injecting dsRNA into adult females (maternal RNAi) resulted in the production of unhatched eggs despite the absence of deformity or fragility of the eggshell. These results suggested that MCO2 plays an important role in sclerotization and pigmentation of the cuticle but not in eggshell integrity in P. stali. Maternal RNAi of any of the other 6 MCO genes and 3 tyrosinase genes affected neither survival nor eggshell integrity of P. stali. Contrary to the observations in the red flour beetle and the brown rice planthopper, RNAi knockdown of MCO6 (MCORP; Multicopper oxidase related protein) exhibited no lethal effects on P. stali. Taken together, our findings provide insight into the functional diversity and commonality of MCOs across hemipteran and other insect groups.

Genetic analysis and molecular detection of resistance to chlorpyrifos mediated by the A216S substitution in acetylcholinesterase-1 in the plant bug Apolygus lucorum.

The green plant bug Apolygus lucorum is a major pest of Bacillus thuringiensis cotton in China. Previously, we reported that chlorpyrifos resistance in a laboratory-selected strain of A. lucorum (BZ-R) is associated with the homozygosis of an allele in the ace-1 gene encoding an alanine to serine substitution at position 216 of acetylcholinesterase-1. Here we describe the results of crosses between the resistant BZ-R strain (41-fold to chlorpyrifos) and the unselected susceptible BZ-S strain homozygous for the wild type alanine allele at position 216. Resistance to chlorpyrifos was inherited as a semi-dominant trait mainly controlled by a single autosomal gene and co-segregates strongly but not completely with the serine substitution in ace-1. Synergism bioassays and enzyme assays showed that minor contributions to resistance are also made by enhanced cytochrome P450 and carboxylesterase activities. A survey of 25 field populations from five Chinese provinces showed strong positive correlations between 50% lethal concentration against chlorpyrifos and S216 allele and genotype frequencies, although the most tolerant populations still only show 40%-50% S216 allele frequencies. The results above provide important information for designing effective resistance monitoring and management strategies for A. lucorum in China.

Proteases and nucleases across midgut tissues of Nezara viridula (Hemiptera:Pentatomidae) display distinct activity profiles that are conserved through life stages.

The southern green stink bug, Nezara viridula is a polyphagous pest of commercially important crops during both nymph and adult stages. This insect has recently transitioned from a secondary agricultural pest to one of primary concern. Novel management solutions are needed due to the limited effectiveness of current control strategies. We performed biochemical and transcriptomic analyses to characterize digestive enzymes in the salivary glands and along midgut tissues of N. viridula nymphs and adults fed on sweet corn. The digestive profiles were more distinct between midgut regions (M1 to M3) than between life stages. Aminopeptidase and chymotrypsin activities declined from the M1 (anterior) toward the M3 midgut region. Cysteine protease activity was higher in the M2 and M3 regions than in M1. Differences in sensitivity to chymotrypsin inhibitors between midgut regions suggest that distinct genes or isoforms are expressed in different regions of the gut. In nymphs, DNA and RNA degradation was higher in M1 than in M3. Adult nuclease activity was low across all midgut regions, but high in salivary glands. The differences in protease activities are reflected by transcriptomic data and functional enrichment of GO terms. Together, our results show that different regions of the digestive tract of N. viridula have specific and distinct digestive properties, and increase our understanding of the physiology of this organism.

An IDS-Type Sesquiterpene Synthase Produces the Pheromone Precursor (Z)-α-Bisabolene in Nezara viridula.

Insects use a wide range of structurally diverse pheromones for intra-specific communication. Compounds in the class of terpenes are emitted as sex, aggregation, alarm, or trail pheromones. Despite the common occurrence of terpene pheromones in different insect lineages, their origin from dietary host plant precursors or de novo biosynthetic pathways often remains unknown. Several stink bugs (Hemiptera: Pentatomidae) release bisabolene-type sesquiterpenes for aggregation and mating. Here we provide evidence for de novo biosynthesis of the sex pheromone trans-/cis-(Z)-α-bisabolene epoxide of the Southern green stink bug, Nezara viridula. We show that an enzyme (NvTPS) related to isoprenyl diphosphate synthases (IDSs) of the core terpene metabolic pathway functions as a terpene synthase (TPS), which converts the general intermediate (E,E)-farnesyl diphosphate (FPP) to the putative pheromone precursor (+)-(S,Z)-α-bisabolene in vitro and in protein lysates. A second identified IDS-type protein (NvFPPS) makes the TPS substrate (E,E)-FPP and functions as a bona fide FPP synthase. NvTPS is highly expressed in male epidermal tissue associated with the cuticle of ventral sternites, which is in agreement with the male specific release of the pheromone from glandular cells in this tissue. Our study supports findings of the function of similar TPS enzymes in the biosynthesis of aggregation pheromones from the pine engraver beetle Ips pini, the striped flea beetle Phyllotreta striolata, and the harlequin bug Murgantia histrionica, and hence provides growing evidence for the evolution of terpene de novo biosynthesis by IDS-type TPS families in insects.

Tissue-specific transcription of proteases and nucleases across the accessory salivary gland, principal salivary gland and gut of Nezara viridula.

The phytophagous stink bug, Nezara viridula (L.) infests multiple plant species and impacts agricultural production worldwide. We analyzed the transcriptomes of N. viridula accessory salivary gland (ASG), principal salivary gland (PSG) and gut, with a focus on putative digestive proteases and nucleases that present a primary obstacle for the stability of protein- or nucleic acid-based stink bug control approaches. We performed high throughput Illumina sequencing followed by de novo transcriptome assemblies. We identified the sequences of 141 unique proteases and 134 nucleases from the N. viridula transcriptomes. Analysis of relative transcript abundance in conjunction with previously reported proteome data (Lomate and Bonning, 2016) supports high levels of serine protease expression in the salivary glands and high cysteine protease expression in the gut. Specifically, trypsin and chymotrypsin transcripts were abundant in the PSG, and cathepsin L-like cysteine protease transcripts were abundant in the gut. Nuclease transcript levels were generally lower than those of the proteases, the exception being abundant transcripts of ribonuclease-C20 in the PSG. The abundance of chymotrypsin, trypsin, and some carboxypeptidase transcripts suggests a significant role for the PSG in production of digestive enzymes. This result is at odds with the premise that the ASG produces watery saliva, which is high in enzymatic activity, while the PSG produces only sheath saliva. We have generated a comprehensive transcriptome sequence dataset from the digestive organs of N. viridula, identified major protease and nuclease genes and confirmed expression of the most abundant enzymes thereby providing greater insight into the digestive physiology of N. viridula.

De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.

Overexpression of cytochrome P450s in a lambda-cyhalothrin resistant population of Apolygus lucorum (Meyer-Dür).

The mirid bug, Apolygus lucorum Meyer-Dür, has been an important pest of cotton crop in China, and is primarily controlled with insecticides, such as pyrethroids. To elucidate the potential resistant mechanisms of A. lucorum to lambda-cyhalothrin, a series of biological, biochemical, and molecular assays were conducted in the reference (AL-S) and lambda-cyhalothrin-resistant (AL-R) populations. Comparison of the molecular target of pyrethroid insecticides, voltage-gated sodium channel, revealed that there were no mutation sites in the resistant population, indicating target insensitivity is not responsible for increased resistance of AL-R to lambda-cyhalothrin. Furthermore, the synergism assays and the activities of detoxification enzymes were performed to determine detoxification mechanism conferring the lambda-cyhalothrin resistance. In the tested synergists, the piperonyl butoxide had the highest synergism ratio against lambda-cyhalothrin, which was up to five-fold in both populations. In addition, the result also showed that only cytochrome P450 had significantly higher O-deethylase activity with 7-ethoxycoumarin (1.78-fold) in AL-R population compared with AL-S population. Seven cytochrome P450 genes were found to be significantly overexpressed in the resistant AL-R population compared with AL-S population. Taken together, these results demonstrate that multiple over-transcribed cytochrome P450 genes would be involved in the development of lambda-cyhalothrin resistance in AL-R population.

Development of a peptide substrate for detection of sunn pest damage in wheat flour.

BACKGROUND: Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeated sequences of high molecular weight glutenin subunits were synthesized. RESULTS: Hydrolysis of peptides by pest protease was determined by high-performance liquid chromatography. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer. The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the assay developed was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants, which are proportional to the protease content of the flour, were determined. CONCLUSION: The total analysis time for the assay developed is estimated as 15 min. The assay developed permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. © 2018 Society of Chemical Industry.

The function and evolutionary significance of a triplicated Na,K-ATPase gene in a toxin-specialized insect.

BACKGROUND: The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell's resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing cardenolides. Here we test the function of these duplicated gene copies in the cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C. RESULTS: Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion, and when we injected either α1A or α1B knockdowns with cardenolides this proved fatal (whereas not in controls). CONCLUSIONS: These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge.

A transgenic strategy for controlling plant bugs (Adelphocoris suturalis) through expression of double-stranded RNA homologous to fatty acyl-coenzyme A reductase in cotton.

Plant bugs (Miridae species), which are sap-sucking insects, have emerged as major pests of cotton in China. Most Miridae species are not sensitive to commercial Bacillus thuringiensis (Bt) cotton, resulting in significant economic losses and an increased application of insecticide, which eventually may compromise the future of Bt cotton. We demonstrate that FATTY ACYL-COA REDUCTASE (AsFAR) plays an essential role in the reproduction of the bug Adelphocoris suturalis. Down-regulation of AsFAR expression by injection of double-stranded RNA suppresses ovarian development and female fertility, resulting in females producing few viable offspring. To determine the viability of an RNA interference approach to limit FAR expression and reproductive ability in A. suturalis, a dsRNA targeting the AsFAR gene (dsAsFAR) of A. suturalis was expressed in transgenic cotton plants. AsFAR transcription levels were significantly downregulated in A. suturalis feeding on the transgenic plants. In contained field trials, the transgenic cotton lines significantly suppressed the development of A. suturalis populations and were resistant to damage caused by plant bug infestation. These results suggest a new strategy for the management of plant bug pests of cotton.

Functional analysis of two polygalacturonase genes in Apolygus lucorum associated with eliciting plant injury using RNA interference.

Salivary enzymes of many piercing-sucking insects lead to host plant injury. The salivary enzymes, polygalacturonase (PGs), act in insect feeding. PG family genes have been cloned from the mirid bug Apolygus lucorum, a pest of cotton and other host crops in China. We investigated the function of two PG genes that are highly expressed in A. lucorum nymphs (PG3-4) and adults (PG3-5), using siRNA injection-based RNA interference (RNAi). Accumulation of mRNA encoding both genes and their cognate proteins was significantly reduced (>60%) in experimental compared control green fluorescent protein (GFP) siRNA-treated mirids at 48 h post injection. Injury levels of cotton buds were also significantly reduced after injecting saliva isolated from PG3-4 and PG3-5 siRNA-treated A. lucorum. These results demonstrate that these two PG act in A. lucorum elicitation of plant injury.

Species diversity can be overestimated by a fixed empirical threshold: insights from DNA barcoding of the genus Cletus (Hemiptera: Coreidae) and the meta-analysis of COI data from previous phylogeographical studies.

The use of genetic distances to identify species within the framework of DNA barcoding has to some extent improved the development of biodiversity studies. However, using a fixed empirical threshold to delimit species may lead to overestimating species diversity. In this study, we use a new data set of COI sequences for 366 specimens within the genus of Cletus as well as conduct an analysis on the same genetic data for collected morphologically defined species from previous phylogeographical studies, to test whether high intraspecific genetic divergences are common with the premises of comprehensive sampling. The results indicate C. graminis Hsiao & Cheng , is the same species with C. punctiger (Dallas, 1852) and should be synonymized and that the distributional record of C. pugnator (Fabricius, 1787) in China is correct. High intraspecific genetic differentiations (0%-4.35%) were found in C. punctiger. Furthermore, as to the mined data, the maximum intraspecific K2P distances of 186 species (48.44% of 384) exceed 3%, and 101 species (26.30%) can be divided into two or more clusters with a threshold of 3% in cluster analysis. If genetic distance is used to delimit species boundaries, the minimum interspecific K2P distance of the congeneric species should be considered rather than only using the fixed empirical value; otherwise, the species richness may be overestimated in some cases.

Role of cathepsins D in the midgut of Dysdercus peruvianus.

Hemipteran ancestors probably lost their digestive serine peptidases on adapting to a plant sap diet. On returning to protein ingestion, these insects start using cathepsin (lysosomal) peptidases as digestive enzymes, from which the less known is cathepsin D. Nine of the ten cathepsin D transcribing genes found in Dysdercus peruvianus midgut are expressed exclusively in this tissue and only DpCatD10 is also expressed in other tissues. The main action of cathepsins D is in the first (V1) (from three, V1-3) midgut regions, where 40% of the total proteolytic activity was assigned to aspartic peptidases with an optimum pH of 3.5. The most expressed cathepsins D were identified in the midgut luminal contents by proteomics. The data indicate that D. peruvianus have kept a lysosomal gene expressed in all tissues and evolved another set of genes with a digestive function restricted to midgut. Digestive cathepsins D apparently complement the action of digestive cathepsin L and they are arguably responsible for the hydrolysis of cysteine peptidase inhibitors known to be present in the cotton seeds eaten by the insect, before they meet cathepsin L.

Gene duplications circumvent trade-offs in enzyme function: Insect adaptation to toxic host plants.

Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K-ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K-ATPase α-subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade-offs in gene function, we expressed the cardenolide-sensitive Na,K-ATPase of Drosophila melanogaster in vitro and introduced four distinct combinations of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K-ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K-ATPase α1C copy maintained activity similar to the wild-type enzyme. Our results suggest that the Na,K-ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier.