Potential Target For Pest Control Research Articles

A C-type lectin with dual-CRD from Tribolium castaneum is induced in response to bacterial challenge.

C-type lectins (CTLs), a group of pattern recognition receptors, are involved in regulating the immune response of insects and could be used as potential targets for pest control. However, information about roles of CTLs in the innate immunity of Tribolium castaneum, a serious, worldwide pest that damages stored grain products, is relatively scarce. Here, a CTL with dual carbohydrate recognition domains (CRDs) containing a highly conserved WHD (Trp53 -His54 -Asp55 ) motif was identified in T. castaneum and named as TcCTL3. Spatiotemporal analysis showed that TcCTL3 was highly expressed in all developmental stages except early eggs, and mainly distributed in central nervous system and hemolymph. The transcript levels of TcCTL3 were significantly increased after lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulation. Recombinant TcCTL3 was able to bind directly to LPS, PGN and all tested bacteria and induce a broad spectrum of microbial agglutination in the presence of Ca2+ . The binding was shown mainly through CRD1 domain of TcCTL3. When TcCTL3 was knocked down by RNA interference, expression of nine antimicrobial peptides (AMPs) (attacin1, attacin2, attacin3, defensins1, defensins2, coleoptericin1, coleoptericin2, cecropins2 and cecropins3) and four transcription factors (TFs) (dif1, dif2, relish and jnk) were significantly decreased under LPS and PGN stimulation, leading to increased mortality of T. castaneum when infected with Gram-positive Staphylococcus aureus or Gram-negative Escherichia coli infection. TcCTL3 could mediate the immune response in T. castaneum via the pattern recognition, agglutination and AMP expression. These findings indicate a potential mechanism of TcCTL3 in resisting bacteria and provide an alternative molecular target for pest control. © 2020 Society of Chemical Industry.

Inhibitor of apoptosis-1 gene as a potential target for pest control and its involvement in immune regulation during fungal infection.

Environmentally friendly insect management technologies, including RNA interference (RNAi) and entomopathogenic fungi, have attracted increasing attention as options for pest control. Here, we sought to extend RNAi-directed targeting of the inhibitor of apoptosis protein 1 (IAP1) gene to the locust, and to examine its relationship to immune responses and susceptibility to Metarhizium acridum, a locust-specific fungal pathogen. Expression of the locust LmIAP gene was induced in the hemolymph and fat body after M. acridum infection. RNAi-directed silencing of locust LmIAP1 resulted in increased caspase 3 activity, degeneration of the gut and dose-dependent mortality. Synergistic mortality was seen in RNAi-LmIAP/fungal co-infection experiments with median survival time (MST) values decreasing from ∼ 5 days for RNAi and M. acridum treatments alone, to 2.6 days for co-treatments. Reduced hemocyte numbers and antimicrobial peptide levels were seen in co-treated locusts, with changes in gut opportunistic pathogenic bacteria seen between treatments. Enhanced fungal sporulation on co-treated insect cadavers was also compared with fungal infection alone. Silencing of the locust LmIAP1 gene results in direct mortality and increases insect susceptibility to insect fungal pathogens, in part by decreasing immunity and altering the gut microbiome. © 2019 Society of Chemical Industry.

Enzymes in the juvenile hormone biosynthetic pathway can be potential targets for pest control.

Discovery of novel insecticides and targets has received global attention in recent years. Ten genes coding for enzymes involved in the juvenile hormone biosynthetic pathway of Manduca sexta were studied as potential insecticide targets. We determined the expression of genes encoding some critical enzymes in the JH biosynthetic pathway. Farnesol dehydrogenase (FOLD), Juvenile hormone acid O-methyltransferase (JHAMT) and Juvenile hormone epoxidase (CYP15C1) were selected as the candidate targets based on gene expression results. RNAi silencing and enzyme inhibitor tests were performed to validate whether these candidate genes could be the potential insecticide targets. The down-regulation of FOLD, JHAMT and CYP15C1 resulted in a 68%, 82% and 79% reduction in the rates of JH biosynthesis in vitro, respectively. In addition, RNA interference and inhibitor studies of these enzymes following oral administration demonstrated the potential application in pest management, with respect to high mortality and effects on growth. Based on our study, FOLD, JHAMT and CYP15C1 could be potential targets for pest control as a consequence of their important roles in insect development. © 2019 Society of Chemical Industry.

Molecular characterization and functional analysis of the vitellogenin receptor in the rice stem borer, Chilo suppressalis.

As a member of the low-density lipoprotein receptor (LDLR) superfamily, vitellogenin (Vg) receptor (VgR) is responsible for the uptake of Vg into developing oocytes and is a potential target for pest control. Here, a full-length VgR complementary DNA (named as CsVgR) was isolated and characterized in the rice stem borer, Chilo suppressalis. The composite CsVgR gene contained an open reading frame of 5,484 bp encoding a protein of 1,827 amino acid residues. Structural analysis revealed that CsVgR contained two ligand-binding domains (LBDs) with four Class A (LDLRA ) repeats in LBD1 and seven in LBD2, which was structurally different from most non-Lepidopteran insect VgRs having five repeats in LBD1 and eight in LBD2. The developmental expression analysis showed that CsVgR messenger RNA expression was first detectable in 3-day-old pupae, sharply increased in newly emerged female adults, and reached a peak in 2-day-old female adults. Consistent with most other insects VgRs, CsVgR was exclusively expressed in the ovary. Notably, injection of dsCsVgR into late pupae resulted in fewer follicles in the ovarioles as well as reduced fecundity, suggesting a critical role of CsVgR in female reproduction. These results may contribute to the development of RNA interference-mediated disruption of reproduction as a control strategy of C. suppressalis.

The functional difference of eight chitinase genes between male and female of the cotton mealybug, Phenacoccus solenopsis.

The cotton mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a polyphagous insect that attacks tens of plant and causes substantial economic loss. Insect chitinases are required to remove the old cuticle to allow for continued growth and development. Though insect chitinases have been well studied in tens of insects, their functions in mealybug are still not addressed. Here, we sequenced the transcriptomes of adult males and females, from which eight chitinase genes were identified. We then used the method of rapid amplification of cDNA ends to amplify their full length. Phylogenetic analysis indicated that these genes clustered into five subgroups. Among which, groupII PsCht2 had the longest transcript and was highly expressed at second instar nymph. PsCht10, PsCht3-3 and PsIDGF were highly expressed in the adult females, whereas PsCht4 and PsCht4-1 were significantly expressed at the male pupa and adult male. Next, we knocked down all eight chitinase genes by feeding the double-stranded RNA. Knockdown of PsCht4 or PsCht4-1 led to the failure of moult and, silencing PsCht5 resulted in pupation defect, while silencing PsCht10 led to small body size, suggesting these genes have essential roles in development and can be used as a potential target for pest control.

Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory, and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models. All data are available on https://citrusgreening.org/.

OUP accepted manuscript

The citrus leaf beetle, Clitea metallica, is a specialized citrus pest through feeding on fresh leaves by larva and adults, and causes nicks and holes into leaves, leaving only a waxy surface layer. Insect cuticle is a complex exoskeleton that is not only involved in development but also protects the insect from environmental contaminations. Due to these key roles of the cuticle, cuticle-related genes are currently investigated in understanding the insect physiology in adaptation. Therefore, in this study, we built two libraries, transcriptomic (43 million clean reads) and small RNA (17 million clean reads), of C. metallica to identify cuticle-related genes and possibly associated miRNAs, being as an example to explore these data sets. Our results showed that a total of 47 cuticular protein genes were identified and most of these genes harbored a conserved motif (the Rebers and Riddiford motif) and belonged to the CPR family. Unigenes encoding proteins involved in chitin synthesis and degradation were also identified, including chitin synthase (2 unigenes), chitinase (14 unigenes), chitinase-like protein (2 unigenes), and chitin deacetylase (5 unigenes). Based on the small RNA library, we identified 30 miRNAs conserved across insect species. Among these miRNAs, 14 were predicted to be target genes associated with cuticle synthesis and degradation. In summary, 70 cuticle-related genes and 14 cuticle-related miRNAs were identified based on the transcriptome and small RNA library of C. metallica. These data sets will promote the understanding of cuticle molecular regulation in C. metallica as well as provide new potential targets for pest control.

Genome-Wide Screening and Functional Analysis Reveal That the Specific microRNA nlu-miR-173 Regulates Molting by Targeting Ftz-F1 in Nilaparvata lugens

Background: Molting is a crucial physiological behavior during arthropod growth. In the past few years, molting as well as chitin biosynthesis triggered by molting, is subject to regulation by miRNAs. However, how many miRNAs are involved in insect molting at the genome-wide level remains unknown.Results: We deeply sequenced four samples obtained from nymphs at the 2nd−3rd and 4th−5th instars, and then identified 61 miRNAs conserved in the Arthropoda and 326 putative novel miRNAs in the brown planthopper Nilaparvata lugens, a fearful pest of rice. A total of 36 mature miRNAs with significant different expression levels at the genome scale during molting, including 19 conserved and 17 putative novel miRNAs were identified. After comparing the expression profiles, we found that most of the targets of 36 miRNAs showing significantly differential expression were involved in energy and hormone pathways. One of the 17 putative novel miRNAs, nlu-miR-173 was chosen for functional study. nlu-miR-173 acts in 20-hydroxyecdysone signaling through its direct target, N. lugens Ftz-F1(NlFtz-F1), a transcription factor. Furthermore, we found that the transcription of nlu-miR-173 was promoted by Broad-Complex (BR-C), suggesting that its involvement in the 20-hydroxyecdysone pathway contributes to proper molting function.Conclusion: We provided a comprehensive resource of miRNAs associated with insect molting and identified a novel miRNA as a potential target for pest control.

The histone deacetylase NlHDAC1 regulates both female and male fertility in the brown planthopper, Nilaparvata lugens.

Histone acetylation is a specific type of chromatin modification that serves as a key regulatory mechanism for many cellular processes in mammals. However, little is known about its biological function in invertebrates. Here, we identified 12 members of histone deacetylases (NlHDACs) in the brown planthopper (BPH), Nilaparvata lugens. RNAi-mediated silencing assay showed that NlHdac1, NlHdac3 and NlHdac4 played critical roles in female fertility via regulating ovary maturation or ovipositor development. Silencing of NlHdac1 substantially increased acetylation level of histones H3 and H4 in ovaries, indicating NlHDAC1 is the main histone deacetylase in ovaries of BPH. RNA sequencing (RNA-seq) analysis showed that knockdown of NlHdac1 impaired ovary development via multiple signalling pathways including the TOR pathway. Acoustic recording showed that males with NlHdac1 knockdown failed to make courtship songs, and thus were unacceptable to wild-type females, resulting in unfertilized eggs. Competition mating assay showed that wild-type females overwhelmingly preferred to mate with control males over NlHdac1-knockdown males. These findings improve our understanding of reproductive strategies controlled by HDACs in insects and provide a potential target for pest control.

Cloning, localization and bioinformatics analysis of a gene encoding an odorant-binding protein (OBP) in Anoplophora glabripennis (Motschulsky).

Anoplophora glabripennis (Motschulsky) has an advanced and complicated olfactory system to identify hosts, mates and spawning locations, and odorant-binding proteins (OBPs) play a key role by binding to volatile materials from different hosts. The full-length cDNA sequence of an OBP, AglaOBP, was cloned by RACE from an antenna cDNA library, and the protein structure and function were predicted by bioinformatics analysis. Gene temporal and spatial expression was detected by real-time qPCR. AglaOBP had distinctive sequence, location and expression profiles compared with other OBPs of A. glabripennis, as it was found in different tissues, and the highest expression was in the elytrums. The possible physiological functions of this OBP were discussed. These findings help elucidate the physiology of this pest and provide a new potential target for pest control.

3-Hydroxy-3-methyl glutaryl coenzyme A reductase is required for ovarian development in the oriental fruit fly Bactrocera dorsalis (Hendel)

Abstract 3-Hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is an important rate-limiting enzyme in the mevalonate pathway, which is critical for the synthesis of juvenile hormone and insect development. In this study, a full-length cDNA encoding HMGR (BdHMGR) was cloned from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frame of BdHMGR encoded 928 amino acids and shared a high degree of identity with other known HMGRs. The expression levels of BdHMGR were high during the early and lately pupal stages, and were low at the larval and mid pupal stages. In the adult stages, the highest level of BdHMGR was detected on day-10 of the adult lifecycle, and the expression levels in females were greater than those in males. The highest tissue-specific expression was in the ovary, followed by fat body and other tissues. Injection of double-stranded RNA (dsRNA) of BdHMGR into adult females significantly reduced BdHMGR transcript levels, and inhibited ovarian development and affected the expression of two yolk protein genes. After dissection, we found that one or two of the spheroids of ovaries from the BdHMGR dsRNA-injected flies were amorphous and reduced in size as compared with the controls. The results suggest that BdHMGR is required for ovarian development in B. dorsalis and may be a potential target for pest control.

Functional characterization of chitin deacetylase 1 gene disrupting larval-pupal transition in the drugstore beetle using RNA interference.

Chitin deacetylases (CDAs) are chitin degradation enzymes that strictly regulate growth and development in insects. In this study, we identified and characterized a full-length cDNA of the CDA gene (SpCDA1) in the drugstore beetle, Stegobium paniceum. The open reading frame of SpCDA1 (1614 bp) encoded a 537 amino acid protein, which possessed typical domain structures of CDAs. Phylogenetic comparison to other insect CDAs revealed that SpCDA1 belongs to Group Ib CDAs. Quantitative real-time PCR analyses showed that SpCDA1 was highly expressed in late larval stages. Significant increase of SpCDA1 transcript level in the larvae was observed upon the exposure of 20-hydroxyecdysone. Injection of double-stranded RNA (dsRNA) of SpCDA1 into the late larvae significantly reduced SpCDA1 transcript levels, resulted in larval-pupal molting difficulty and produced high larval mortality. After 15 days, the survival rate of S. paniceum in dsSpCDA1 group was significantly reduced by 72% compared to the control. The results demonstrated that SpCDA1 is essential for successful larval-pupal transition in S. paniceum and this gene may be a potential target for pest control.

Differential accumulation of leucine and methionine in red and green pea aphids leads to different fecundity in response to nitrogen fertilization.

Nitrogen fertilization affects plants directly and herbivorous insects indirectly. Although insect species and even genotypes are known to differ in their responses to nitrogen fertilization, the physiological and molecular mechanisms remain unclear. This study assessed the fecundity and related regulatory signaling pathways in the green and red morphs of pea aphid (Acyrthosiphon pisum) feeding on Medicago truncatula with and without nitrogen fertilization. Nitrogen fertilization significantly increased foliar amino acid concentrations and consequently increased the concentrations of several individual essential amino acids in body tissue of the green morph. The increased concentration of Leu, Ile, Met and Val was consistent with enhanced biosynthesis of these amino acids in the endosymbiont Buchnera. Under nitrogen fertilization, Leu and Met accumulated in the green morph enhanced the target of rapamycin (TOR) signaling pathway, which consequently increased fecundity by promoting vitellogenin synthesis. In the red morph, however, nitrogen fertilization did not change the concentration of essential amino acids, TOR signaling or fecundity. Specific amino acids accumulation and the nutrient transduction pathway in pea aphids are responsible for genotype-specific fecundity in response to nitrogen fertilization, which could be used as potential target for pest control. © 2018 Society of Chemical Industry.

Identification of a sugar gustatory receptor and its effect on fecundity of the brown planthopper Nilaparvata lugens.

In insects, the gustatory system plays a crucial role in multiple physiological behaviors, including feeding, toxin avoidance, courtship, mating and oviposition. Gustatory stimuli from the environment are recognized by gustatory receptors. To date, little is known about the function of gustatory receptors in agricultural pest insects. In this study, we cloned a sugar gustatory receptor gene, NlGr11, from the brown planthopper (BPH), Nilaparvata lugens (Stål), a serious pest of rice in Asia; we then identified its ligands, namely, fructose, galactose and arabinose, by calcium imaging assay. After injection of NlGr11 double-stranded RNA, we found that the number of eggs laid by BPH decreased. Moreover, we found that NlGr11 inhibited the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and promoted the phosphorylation of protein kinase B (AKT). These findings demonstrated that NlGr11 could accelerate the fecundity of BPH through AMPK- and AKT-mediated signaling pathways. This is the first report to indicate that a gustatory receptor modulates the fecundity of insects and that the receptor could be a potential target for pest control.

Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.

Vitellogenin Receptor Mutation Leads to the Oogenesis Mutant Phenotype “scanty vitellin” of the Silkworm, Bombyx mori

The vitellogenin receptor (VgR) mediates the uptake of vitellogenin (Vg) from the hemolymph by developing oocytes. VgR with the mutational EGF1 domain can bind ligand proteins but cannot be dissociated under acidic conditions. The mutant is lethal in embryos. Bombyx mori VgR (BmVgR) has an important role in egg formation and embryonic development. BmVgR is a potential target for pest control. In insects, the vitellogenin receptor (VgR) mediates the uptake of vitellogenin (Vg) from the hemolymph by developing oocytes. The oogenesis mutant scanty vitellin (vit) of Bombyx mori (Bm) lacks vitellin and 30-kDa proteins, but B. mori egg-specific protein and BmVg are normal. The vit eggs are white and smaller compared with the pale yellow eggs of the wild type and are embryonic lethal. This study found that a mutation in the B. mori VgR gene (BmVgR) is responsible for the vit phenotype. We cloned the cDNA sequences encoding WT and vit BmVgR. The functional domains of BmVgR are similar to those of other low-density lipoprotein receptors. When compared with the wild type, a 235-bp genomic sequence in vit BmVgR is substituted for a 7-bp sequence. This mutation has resulted in a 50-amino acid deletion in the third Class B region of the first epidermal growth factor (EGF1) domain. BmVgR is expressed specifically in oocytes, and the transcriptional level is changed dramatically and consistently with maturation of oocytes during the previtellogenic periods. Linkage analysis confirmed that BmVgR is mutated in the vit mutant. The coimmunoprecipitation assay confirmed that mutated BmVgR is able to bind BmVg but that BmVg cannot be dissociated under acidic conditions. The WT phenotype determined by RNA interference was similar to that of the vit phenotype for nutritional deficiency, such as BmVg and 30-kDa proteins. These results showed that BmVgR has an important role in transporting proteins for egg formation and embryonic development in B. mori.

In silico cloning and annotation of genes involved in the digestion, detoxification and RNA interference mechanism in the midgut of Bactrocera dorsalis [Hendel (Diptera: Tephritidae)

As the second largest organ in insects, the insect midgut is the major tissue involved in the digestion of food and detoxification of xenobiotics, such as insecticides, and the first barrier and target for oral RNA interference (RNAi). In this study, we performed a midgut-specific transcriptome analysis in the oriental fruit fly, Bactrocera dorsalis, an economically important worldwide pest, with many populations showing high levels of insecticide resistance. Using high-throughput sequencing, 52 838 060 short reads were generated and assembled to 25 236 unigenes with a mean length of 758 bp. Interestingly, 34 unique sequences encoding digestion enzymes were newly described and these included aminopeptidase and trypsin, genes associated with Bacillus thuringiensis resistance and fitness cost. Second, 41 transcripts were annotated to particular detoxification genes such as glutathione S-transferases, carboxylesterases and cytochrome P450s, and the subsequent phylogenetic analysis indicated homology with tissue-specific and insecticide resistance-related genes of Drosophila melanogaster. Third, we identified the genes involved in the mechanism of RNAi and the uptake of double-stranded RNA. The sequences encoding Dicer-2, R2D2, AGO2, and Eater were confirmed, but SID and SR-CI were absent in the midgut transcriptome. In conclusion, the results provide basic molecular information to better understand the mechanisms of food digestion, insecticide resistance and oral RNAi in this important pest insect in agriculture. Specific genes in these systems can be used in the future as potential targets for pest control, for instance, with RNAi technology.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase ( Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 °C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and r Sl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC ( k cat /K m = 37.53 mM S −1), which is similar to other insect cathepsin L-like enzymes. r Sl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. r Sl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 ( K i = 0.196 nM), indicating that this protease is a potential target for pest control.

Isolation and Identification of Insect Intestinal Mucin Haiim86 - The New Target for Helicoverpa Armigera Biocontrol

There are many more glycoproteins in Helicoverpa armigera peritrophic membrane than midgut separated by SDS-PAGE analysis after Periodic acid-Schiff (PAS) and coomassie staining. The peritrophic membrane (PM) of H. armigera larvae contains about forty associated proteins. A cDNA library was constructed from H. armigera midgut mRNA to study the new target for pest biocontrol. An antiserum against Spodoptera exigua integral/total PM proteins cross reacted with several H. armigera PM proteins and was used to isolate a complete cDNA encoding an insect intestinal mucin (HaIIM86). The deduced protein sequence of the cDNA contained one potentially glycosylated, mucin-like domain, five cysteine-rich chitin-binding domains (CBDs) and two D-G rich regions. Mucin domain was lined between the first and second CBDs; the two additional D-G rich regions were proposed to internal reside at the amino terminus of the protein flanked by three cysteine-rich CBDs. HaIIM86 contains two D-G-rich tandem repeat domains flanked by cysteine-rich sequences in peritrophic membrane proteins which is not present in all the currently known PM proteins. Howerer the functions of D-G rich domains require further investigation. HaIIM86 was shown as 200 kDa protein by SDS-PAGE analysis and appeared to be associated with the PM. HaIIM86 has chitin-binding activity and can be degraded into 90 and 70 kDa by HaGV Enhancin in vivo. The finding has shown that HaIIM86 is the target substrate for enhancin and the potential target for pest control.