Inhibition of trehalase affects the trehalose and chitin metabolism pathways in Diaphorina citri (Hemiptera: Psyllidae)

The Asian citrus psyllid, Diaphorina citri is the principal vector of huanglongbing, which transmits Candidatus Liberibacter asiaticus. Trehalase is a key enzyme involved in trehalose hydrolysis and plays an important role in insect growth and development. The specific functions of this enzyme in D. citri have not been determined. In this study, three trehalase genes (DcTre1-1, DcTre1-2, and DcTre2) were identified based on the D. citri genome database. Bioinformatic analysis showed that DcTre1-1 and DcTre1-2 are related to soluble trehalase, whereas DcTre2 is associated with membrane-bound trehalase. Spatiotemporal expression analysis indicated that DcTre1-1 and DcTre1-2 had the highest expression levels in the head and wing, respectively, and DcTre2 had high expression levels in the fat body. Furthermore, DcTre1-1 and DcTre1-2 expression levels were induced by 20-hydroxyecdysone and juvenile hormone Ⅲ, but DcTre2 was unaffected. The expression levels of DcTre1-1, DcTre1-2, and DcTre2 were significantly upregulated, which resulted in high mortality after treatment with validamycin. Trehalase activities and glucose contents were downregulated, but the trehalose content increased after treatment with validamycin. In addition, the expression levels of chitin metabolism-related genes significantly decreased at 24 and 48 h after treatment with validamycin. Furthermore, silencing of DcTre1-1, DcTre1-2, and DcTre2 reduced the expression levels of chitin metabolism-related genes and led to a malformed phenotype of D. citri. These results indicate that D. citri trehalase plays an essential role in regulating chitin metabolism and provides a new target for control of D. citri.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

7种内共生病毒对柑橘木虱生殖力的影响

[目的]柑橘木虱吸食柑橘和九里香等芸香科植物,更是柑橘黄龙病的主要传播媒介。了解柑橘木虱共生微生物对虫体产卵量的影响,可为该虫的生态调控提供理论依据。[材料]应用Illumina HiSeq技术对柑橘木虱进行转录组和小RNA (siRNA)高通量测序,对拼接的序列进行功能注释。通过单雌单苗的饲养方法,分析通过测序揭晓的7种内共生病毒对柑橘木虱的影响,包括单雌总产卵量、单雌日产卵量和单雌寿命的变化。[结果]通过高通量测序共获得unigenes序列22429条,共有17673条unigenes注释至NR、NT、Pfam等数据库。根据siRNA分析数据,获得了多条柑橘木虱内共生病毒的序列。生物学验证试验发现,柑橘木虱呼肠孤病毒、Hubei tick virus 2、褐飞虱呼肠孤病毒、Mal de Rio Cuarto virus、Synechococcus phage S-RSM4、Tokyovirus A1 DNA和Diaphorina citri picorna-like virus isolate BR1 7种内共生病毒显著降低了雌虫群体总产卵量,但是对单雌日产卵量和单雌寿命无显著影响。[结论]研究结果为柑橘木虱的共生微生物多样性提供证据,也为进一步探索内共生病毒对宿主昆虫的意义提供了思路和理论基础,为后续柑橘木虱-内共生微生物的互作研究提供数据支持。     

Silencing of V-ATPase-E gene causes midgut apoptosis of Diaphorina citri and affects its acquisition of Huanglongbing pathogen

The Asian citrus psyllid, Diaphorina citri Kuwayama, is among the most important pests of citrus. It is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe losses in citrus crops. Control of D. citri is therefore of paramount importance to reduce the spread of HLB. In this regard, using RNA interference (RNAi) to silence target genes is a useful strategy to control psyllids. In this study, using RNAi, we examined the biological functions of the V-ATPase subunit E (V-ATP-E) gene of D. citri, including its effect on acquisition of CLas. The amino acid sequence of V-ATP-E from D. citri had high homology with proteins from other insects. V-ATP-E was expressed at all D. citri life stages analyzed, and the expression level in mature adults was higher than that of teneral adults. Silencing of V-ATP-E resulted in a significant increase in mortality, reduced body weight, and induced cell apoptosis of the D. citri midgut. The reduced expression of V-ATP-E was indicated to inhibit CLas passing through the midgut and into the hemolymph, leading to a majority of CLas being confined to the midgut. In addition, double-stranded RNA of D. citri V-ATP-E was safe to non-target parasitic wasps. These results suggest that V-ATP-E is an effective RNAi target that can be used in D. citri control to block CLas infection.     

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.     

Cuticular protein LmTwdl1 is involved in molt development of the migratory locust

The cuticle, an essential structure for insects, is produced from cuticular proteins and chitin via a series of biochemical reactions. Tweedle genes are important members of the cuticular protein family and have four conserved motifs binding to chitin. Tweedle family genes have been found to play a profound effect on cuticle development. Here, we report that the cuticular protein gene LmTwdl1 of Locusta migratoria belongs to the Tweedle family. In situ hybridization showed that LmTwdl1 is localized to epidermal cells of the cuticle. The expression patterns of LmTwdl1 showed low expression in the cuticle during the early and middle stages of the fifth‐instar nymphs; in contrast, its expression rapidly increased in the late stages of fifth‐instar nymphs. We performed RNA interference to examine the function of LmTwdl1 in locusts. Silencing of LmTwdl1 resulted in high mortality during the molting process before the next stage. Also, the epicuticle of nymphs failed to molt, tended to be thinner and the arrangement of chitin in the procuticle appeared to be disordered compare to the control group. These results demonstrate that LmTwdl1 plays a critical role in molting, which contributes to a better understanding of the distinct functions of the Tweedle family in locusts.     

Proteomic maps of subcellular protein fractions of the Asian citrus psyllid Diaphorina citri,the vector of citrus huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two‐dimensional gel electrophoresis (2‐DE). A total of 1170 spots are detected in the 2‐DE gels of the three subcellular fractions. One hundred and sixty‐four differentially expressed proteins are successfully identified using liquid chromatography‐dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.     

Double-Stranded RNA Uptake through Topical Application,Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in Diaphorina citri

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P₄₅₀ genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P₄₅₀ proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.     

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

Chitinases: in agriculture and human healthcare

Abstract

Biological control of phytopathogenic fungi and insects continues to inspire the research and development of environmentally friendly bioactive alternatives. Potentially lytic enzymes, chitinases can act as a biocontrol agent against agriculturally important fungi and insects. The cell wall in fungi and protective covers, i.e. cuticle in insects shares a key structural polymer, chitin, a β-1,4-linked N-acetylglucosamine polymer. Therefore, it is advantageous to develop a common biocontrol agent against both of these groups. As chitin is absent in plants and mammals, targeting its metabolism will signify an eco-friendly strategy for the control of agriculturally important fungi and insects but is innocuous to mammals, plants, beneficial insects and other organisms. In addition, development of chitinase transgenic plant varieties probably holds the most promising method for augmenting agricultural crop protection and productivity, when properly integrated into traditional systems. Recently, human proteins with chitinase activity and chitinase-like proteins were identified and established as biomarkers for human diseases. This review covers the recent advances of chitinases as a biocontrol agent and its various applications including preparation of medically important chitooligosaccharides, bioconversion of chitin as well as in implementing chitinases as diagnostic and prognostic markers for numerous diseases and the prospect of their future utilization.     

柑橘内生真菌的分离鉴定及其发酵产物对柑橘溃疡病菌的抑制活性

本研究从柑橘抗病品种的健康植株不同组织中分离纯化和鉴定内生真菌,并测定其发酵产物对柑橘溃疡病菌的抑制活性,以明确柑橘抗病品种中内生真菌的组成及其产抗柑橘溃疡病菌活性代谢产物的潜力,为柑橘溃疡病抗菌剂的开发奠定基础。该研究通过组织培养法分离内生真菌,采用形态学和分子生物学方法对其进行鉴定; 基于前期的拮抗预试验结果,选取代表性菌株进行发酵培养,通过乙酸乙酯浸提、真空抽滤、旋转蒸发浓缩制备粗提物; 采用带毒平板涂布法测定不同菌株发酵产物乙酸乙酯提取物对柑橘溃疡病菌的抑制活性。结果表明:(1)共分离得到72株内生真菌,归为2门(Ascomycota、Basidiomycota)、14个属,其中优势属为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、链格孢属(Alternaria)和镰刀菌属(Fusarium)。(2)不同柑橘品种中内生真菌多样性指数为温州蜜柑(桂林)>沙糖桔(桂林)>沙糖桔(梧州)。(3)不同组织中内生真菌多样性变化因地理位置差异而有所不同,采自桂林的温州蜜柑和沙糖桔均为叶片中的内生真菌的多样性高于枝条,而采自梧州的沙糖桔为叶片中的多样性低于枝条,并且采自梧州的柑橘样品与采自桂林的柑橘样品中的内生真菌相似性低。(4)测定了30株内生真菌乙酸乙酯提取物对柑橘溃疡病菌的抑制活性,其中29株菌株表现出不同程度抑制活性。不同柑橘品种中的优势属的MIC介于0.312 5～10 mg·mL^-1之间,特有属的MIC介于0.156～5 mg·mL^-1,共有属镰刀菌属的MIC介于0.312 5～2.5 mg·mL^-1之间。研究结果表明柑橘抗病品种中内生真菌具有丰富多样性,并且其发酵提取物普遍对柑橘溃疡病菌具有抑制作用。特有属抑菌活性总体优于优势属,共有属镰刀菌属在不同柑橘抗病品种中均具有显著抑菌效果。     

Inhibition of fatty acid desaturation impairs cuticle differentiation in Drosophila melanogaster

Previously, we showed that inhibition of the activity of fatty acid desaturases (Desat) perturbs signalling of the developmental timing hormone ecdysone in the fruit fly Drosophila melanogaster. To understand the impact of this effect on cuticle differentiation, a process regulated by ecdysone, we analysed the cuticle of D. melanogaster larvae fed with the Desat inhibitor CA10556. In these larvae, the expression of most of the key cuticle genes is normal or slightly elevated at day one of CA10556 feeding. As an exception, expression of twdlM coding for a yet uncharacterised cuticle protein is completely suppressed. The cuticle of these larvae appears to be normal at the morphological level. However, these animals are sensitive to desiccation, a trait that according to our data, among others, may be associated with reduced TwdlM amounts. At day two of CA10556 feeding, expression of most of the cuticle genes tested including twdlM is suppressed. Expression of cpr47Eb coding for a chitin‐binding protein is, by contrast, highly elevated suggesting that Cpr47Eb participates at a specific compensation program. Overall, the cuticle of these larvae is thinner than the cuticle of control larvae. Taken together, lipid desaturation is necessary for a coordinated deployment of a normal cuticle differentiation program.     

Comparative proteomic analysis between fifth‐instar nymphs and adults of Asian citrus psyllid Diaphorina citri

Asian citrus psyllid Diaphorina citri Kuwayama is extremely problematic worldwide, particularly where Huanglongbing (HLB) disease, the most serious and devastating of citrus diseases, is found. The threat is a result of its ability to transmit the causal agent of HLB, Candidatus Liberibacter asiaticus (CLas) bacterium. Improvements in proteomics, mass spectrometry, bioinformatics tools and gene ontology annotation facilitate the mapping and large‐scale identification and quantification of proteins. To date, only a few comparative proteomic studies report the developmental proteomic changes of hemimetabolous and plant–disease vector insects. Two‐dimensional gel electrophoresis analysis of D. citri total protein is able to detect qualitative and quantitative developmental differences. Liquid chromatography‐tandem mass spectrometry identifies 89 protein spots. Most proteins are metabolism and bioenergetics‐related. Nineteen protein spots are found to be implicated in stress/defence/immunity; 7 in development regulation; 9 in nervous system functions; 4 in the reproductive system; 23 in cytoskeleton and muscle organization; and 4 in movement, flight and other processes. Significant increases in the level of proteins related to structural constitution of the skeleton, stress/defence/immunity, reproduction system, muscles, locomotion and flight are found in adults, consistent with the fact that D. citri is a hemimetabolous insect, whereas proteins involved in developmental regulation are higher in the nymphal stage. The identification of these variably expressed proteins between the nymph and adult stages, linked with the basis of their physiological roles, will lead to a better understanding of the factors influencing development in D. citri and the regulation of some crucial metabolic pathways. It may also help to identify targets for genetic manipulation using RNA interference or other techniques to disrupt Asian citrus psyllid development, lifespan or its ability to transmit CLas.