Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.     

Chitin synthases are required for survival, fecundity and egg hatch in the red flour beetle, Tribolium castaneum

The synthesis of chitin, the beta-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, TcCHS-A and TcCHS-B, during embryonic and adult development in the red flour beetle, Tribolium castaneum, RNA interference (RNAi) experiments were carried out. When dsRNA for TcCHS-A was injected into male or female pharate adults, all insects died 5-7 d after the adult molt, and the females failed to oviposit prior to death. When dsTcCHS-A was injected into young adults 1-2 d post-eclosion, a similar lethal phenotype was obtained after 5 d and no oviposition occurred. When dsTcCHS-A injections were delayed until after adult maturation (7-10 d post-eclosion), the treated females did oviposit and the resulting embryos appeared to develop normally. However, the chitin content of the eggs was dramatically reduced, the embryos became twisted and enlarged, and the eggs did not hatch. Adults treated with dsRNA for TcCHS-B exhibited little or no chitin in their PM and died about 2 wk after injection. None of the TcCHS-B-treated females oviposited, which was probably a secondary effect caused by starvation. These results extend our previous findings that CHS genes are required for all types of molt. The present study also demonstrates that these genes have additional roles in embryonic and adult development.     

Cysteine protease enhances plant-mediated bollworm RNA interference

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665–673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.     

Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.     

Different Functions of the Insect Soluble and Membrane-Bound Trehalase Genes in Chitin Biosynthesis Revealed by RNA Interference

Background

Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported.

Principal Findings

The membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%.

Conclusions

SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut.     

Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae.     

Cloning,characterization, and RNAi effect of the chitin synthase B gene in Cnaphalocrocis medinalis

The rice leaf folder (RLF), Cnaphalocrocis medinalis, is one of the major pests of rice, and chitin synthase is a key enzyme for the chitin synthesis pathway in insects. In this study, the chitin synthase B gene from C. medinalis (CmCHSB) was cloned and characterized. The cDNA of CmCHSB is 4824 bp in length, containing an open reading frame of 4578 nucleotides that encodes 1525 amino acids. The CmCHSB zymoprotein consists of 10 transmembrane domains (TMDs) in the N-terminus, a middle conserved catalytic domain, and 7 TMDs in the C-terminus. Homology and phylogenetic analyses revealed that CmCHSB possesses the closest relationship with its homolog in Ostrinia furnacalis. CmCHSB was expressed throughout development and in all of the adult tissues tested, with the highest expression level in the adult and in the midgut. Silencing of CmCHSB through RNA interference (RNAi) severely affected RLF larval growth and caused larval lethality. Our results revealed that CmCHSB is essential for RLF growth and development, which sheds new light on the characteristics and functions of this gene. These findings will be helpful for green control of RLF, by targeting the CmCHSB gene using RNAi technology.     

Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum

Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level.     

Calcofluor disrupts the midgut defense system in insects

The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.     

A Biochemical Evidence of the Inhibitory Effect of Diflubenzuron on the Metamorphosis of the Silkworm,Bombyx mori

Diflubenzuron (DFB) has been known to prevent metamorphosis of silkworm, Bombyx mori, from larval to pupal stage at low dose exposure. To explain this inhibitory action of DFB, a hypothesis was raised that DFB acts like juvenile hormone (JH) or DFB inhibits JH esterase to increase endogenous JH titer. A JH bioassay using isolated abdomen clearly indicates that DFB does not act as JH analog because DFB did not induce vitellogenesis in the isolated female abdomen, while endogenous JHs did significantly. General esterase activities in hemolymph were lower in DFB-treated fifth instar larvae than in the control larvae, but there was no difference between fat body esterase activities in both groups. Two hemolymph esterases (‘E1’ and ‘E2’) of the fifth instar larvae were separated and visualized by α-and β-naphthyl acetate. From in vitro incubation experiment, the cathodal esterase (‘E1’) was sensitive to DFB at its nanomolar range. Considering the fact that early fifth instar larvae have high level of JH esterase in the hemolymph, these results suggest that DFB inhibit larval to pupal metamorphosis by blocking JH degradation, which increases endogenous JH titer especially at the critical period when the larvae determine metamorphic development at the following molt.     

The intestinal barrier in lepidopteran larvae: permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides

Endogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway. We characterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images of whole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through the transcellular route.     

The origin and functions of the insect peritrophic membrane and peritrophic gel

There is a a fluid (peritrophic gel) or membranous (peritrophic membrane, PM) film surrounding the food bolus in most insects. The PM is composed of chitin and proteins, of which peritrophins are the most important. It is proposed here that, during evolution, midgut cells initially synthesized chitin and peritrophins derived from mucins by acquiring chitin-binding domains, thus permitting the formation of PM. Since PM compartmentalizes the midgut, new physiological roles were added to those of the ancestral mucus (protection against abrasion and microorganism invasion). These new roles are reviewed in the light of data on PM permeability and on enzyme compartmentalization, fluid fluxes, and ultrastructure of the midgut. The importance of the new roles in relation to those of protection is evaluated from data obtained with insects having disrupted PM. Finally, there is growing evidence suggesting that a peritrophic gel occurs when a highly permeable peritrophic structure is necessary or when chitin-binding molecules or chitinase are present in food.     

The effects of DOPA decarboxylase inhibitors on the permeability and ultrastructure of the larval cuticle of the Australian sheep blowfly, Lucilia cuprina

Larvae of Lucilia cuprina, fed toxic levels of α-methyl DOPA (or other DOPA decarboxylase inhibitors) during the first or second instar, die at the completion of the next moult, soon after exposing their new cuticles. In electron micrographs of newly synthesised cuticle from these treated larvae, the ultrastructure of the lipid-rich outer epicuticle layer appears to be abnormal. This newly formed cuticle of the treated larvae is apparently defective in its role as a water permeability barrier (compared with that of normal larvae), since it permits the free movement of water in both directions. Thus, treated larvae die most probably as a direct result of dehydration. Larvae fed toxic levels of α-methyl DOPA can be rescued from death by simultaneously adding N-acetyldopamine (the cuticular sclerotizing agent) to the food. The rescued larvae are apparently normal in all respects. This suggests that sclerotization is required for the formation of a normal outer epicuticle. Diflubenzuron, which is known to inhibit chitin deposition in the cuticles of a number of different species of insect, also apparently affects chitin deposition in the larval cuticle of L. cuprina. Thus, in electron micrographs of cuticle from larvae fed toxic levels of diflubenzuron the ultrastructure of the chitin-containing endocuticle layer appears to be abnormal.