Injection Of dsRNA Research Articles

Knockdown of several components of cytochrome P450 enzyme systems by RNA interference enhances the susceptibility of Helicoverpa armigera to fenvalerate

The function of cytochrome P450 proteins (P450s) in the metabolism of a variety of compounds by oxidation and reduction is well elucidated, but its interactions with other electron transfer components in the pyrethroid resistance of insect pests have been a mystery for a long time. In previous studies the authors cloned and characterised CYP6B7 and cytochrome b(5) (Cyt-b(5)) in the fenvalerate-resistant HDFR strain of cotton bollworm (Helicoverpa armigera Hübner) and showed that CYP6B7 mRNA was overexpressed and important for resistance to fenvalerate. In the present study, the functional interactions of CYP6B7, NADPH-dependent cytochrome P450 reductase (CPR) and Cyt-b(5) were assessed using RNA interference (RNAi) strategies and monitoring for fenvalerate resistance levels. RT-qPCR analyses indicated that the expression levels of CYP6B7, CPR and Cyt-b(5) mRNA were decreased drastically in the midgut of fourth-instar larvae of the H. armigera HDFR strain after corresponding double-stranded RNA (dsRNA) injection, compared with that of the control. The knockdown of CYP6B7, CPR and Cyt-b(5) transcripts was time course dependent during a 12-48 h period after dsRNA injection. At the earlier time points analysed, significant suppression of CYP6B7 mRNA levels was observed in larvae injected with dsCYP6B7-313 as compared with controls, and further suppression was observed in larvae injected with dsCYP6B7-313, dsCPR-403 and dsCyt-b(5) . The injection of dsCYP6B7-313 together with dsCPR-403 and dsCyt-b(5) increased larval susceptibility of the HDFR strain to fenvalerate. The results demonstrated that silencing of CYP6B7 alone or CYP6B7 together with CPR and/or Cyt-b(5) increased the susceptibility of H. armigera to fenvalerate, suggesting that CYP6B7, CPR and Cyt-b(5) collaboratively participated in enhanced metabolism of fenvalerate and played an important role in the resistance of H. armigera to fenvalerate.

Identification, localization and function of glutamate‐gated chloride channel receptors in the honeybee brain

Glutamate-gated chloride channels (GluCls) are members of the cys-loop ligand-gated ion channel superfamily whose presence has been reported in a variety of invertebrate tissues. In the honeybee, a single gene, amel_glucl, encoding a GluClα subunit, was found in the genome but both the pattern of expression of this gene in the bee brain and its functional role remained unknown. Here we localised the expression sites of the honeybee GluClα subunit at the mRNA and protein levels. To characterise the functional role of GluCls in the honeybee brain, we studied their implication in olfactory learning and memory by means of RNA interference (RNAi) against the GluClα subunit. We found that the GluClα subunit is expressed in the muscles, the antennae and the brain of honeybees. Expression of the GluClα protein was necessary for the retrieval of olfactory memories; more specifically, injection of dsRNA or siRNA resulted in a decrease in retention performances ∼24 h after injection. Knockdown of GluClα subunits impaired neither olfaction nor sucrose sensitivity, and did not affect the capacity to associate odor and sucrose. Our data provide the first evidence for the involvement of glutamate-gated chloride channels in olfactory memory in an invertebrate.

Chitin synthase 1 gene and its two alternative splicing variants from two sap-sucking insects, Nilaparvata lugens and Laodelphax striatellus (Hemiptera: Delphacidae)

Chitin synthase (CHS) is an enzyme that is required for chitin formation in insect cuticles and other tissues. In this study, CHS genes from two destructive rice insect pests, the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus, were cloned. Phylogenetic analysis showed that these genes belonged to class CHS1 of the CHS gene family. Most insects possess two CHS genes (CHS1 and CHS2); however, genome and transcriptome searches showed that N. lugens possibly possess only CHS1 in both databases. Two transcript variants (CHS1a and CHS1b) resulting from exclusively alternative splicing (exon 19a or 19b in N. lugens) were identified for each of the two rice planthopper CHS1s. Gene structure comparison using the genomes that are currently sequenced showed that the CHS1 genes in all insects except Acyrthosiphon pisum have two transcript variants. Transcription of NlCHS1a reached its highest level just after molting, whereas NlCHS1b reached its highest expression level 1–2 days before molting. Injection of the N. lugens nymphs with double-strand RNA (dsRNA) of CHS1, CHS1a and CHS1b reduced the corresponding variant transcript levels and exhibited subsequent phenotypes. Silencing of CHS1 and CHS1a resulted in elongated distal wing pads and the “wasp-waisted” or crimpled cuticle phenotypes and eventually died, whereas the phenotypes caused by injection of NlCHS1b dsRNA seem not so obvious although slightly increased mortality was observed. Our results suggest that N. lugens likely lacks CHS2 and CHS1 may be efficient target gene for RNAi-based N. lugens control.

The SID-1 double-stranded RNA transporter is not required for systemic RNAi in the migratory locust

Systemic RNAi, the spreading of RNAi effects to cells and tissues that have not initially encountered a dsRNA trigger, is a common phenomenon in many organisms. However, the underlying mechanisms of systemic RNAi remain largely unknown. Here, we studied the characteristics and possible mechanisms of systemic RNAi in Locusta migratoria. We observed that the locust has pronounced sensitive systemic RNAi in response to dsRNA injection for both broadly-expressed as well as tissue-specific genes. Only a 30 pg (dsRNA / mg tissues) dose could induce significant systemic RNAi effects. Only one SID-1 ortholog (LmSID-1) was identified in the locust genome, which exhibited a progressively increasing expression pattern with development and its expression was enriched in the gonad. To test the role of LmSID-1 in systemic RNAi, we performed in vitro and in vivo experiments. The results from in vivo experiments showed that silencing of LmSID-1 gene dose not influence RNAi effects of other genes. The results from in vitro experiments confirmed that the expression of the LmSID-1 protein in Drosophila S2 cells could not enhance dsRNA uptake. Thus, these findings imply the existence of alternative mechanisms underlying insect systemic RNAi, which may be different from Caenorhabditis elegans or mammals.

Molecular cloning and functional study of calreticulin from a lepidopteran pest, Pieris rapae

Insects have an effective innate immune system to protect themselves from exogenous invaders. Calreticulin is a multifunctional protein mainly involved in directing proper conformation of proteins, controlling calcium level, and participating in immune responses. Previous suppression subtractive hybridization assay showed that the expression of Pieris rapae calreticulin (PrCRT) was suppressed after injection of Pteromalus puparum venom. In this study, we obtained a full length cDNA of PrCRT and expressed recombinant wild type and the N-domain deleted mutant PrCRT in bacteria. Real time quantitative PCR and western blot analyses showed that PrCRT mRNA and protein were expressed in hemocytes, Malpighian tubule, midgut, epidermis and fat body, with a higher level in hemocytes. PrCRT was probably located in endoplasmic reticulum distributing in the cytoplasm of hemocytes. Recombinant PrCRT was first able to attach and then enter the hemocytes by endocytosis. PrCRT mRNA in hemocytes was significantly induced after injection of yeast or beads, but did not change noticeably after injection of Escherichia coli or Micrococcus lysodeikticus. Recombinant PrCRT enhanced cellular encapsulation by P. rapae hemocytes in vitro, and the N-domain of PrCRT was required for encapsulation. RNAi of PrCRT by dsRNA injection impaired the ability of hemocytes to encapsulate beads. After parasitization by P. puparum, PrCRT mRNA and protein levels in P. rapae pupal hemocytes were significantly suppressed compared to non-parasitized control. Our results suggest that PrCRT is involved in cellular encapsulation and the pupal parasitoid P. puparum can decrease PrCRT expression to impair host cellular immune response.

FUNCTIONAL ANALYSIS OF TWO CHITINASE GENES DURING THE PUPATION AND ECLOSION STAGES OF THE BEET ARMYWORM Spodoptera exigua BY RNA INTERFERENCE

Insect chitinases are a multigene family that is encoded by a rather large and diverse group of genes. The main function of chitinases is to digest the chitin contained in tissues such as the cuticles and gut lining during molting. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) during the pupation and eclosion stages of Spodoptera exigua. First, efficient silencing of the SeChi and SeChi-h genes through specific double-stranded RNA (dsRNA) injection led to a significant reduction in the mRNA levels of SeChi and SeChi-h. Additionally, different phenotypic defects were observed at the pupal and adult stages after injection of the SeChi and SeChi-h dsRNAs. After injecting SeChi dsRNA in the pupal stage, the cuticle of the head split open and the pupal cuticle was visible under the old larval cuticle. However, after injecting the SeChi-h dsRNA, animals died without exhibiting any special phenotypes. At the adult death stage, animals injected with dsSeChi could not shed their pupal shell completely, and their old cuticles remained attached to their head or chest. However, the main lethal phenotype was that insects did not emerge after dsSeChi-h injection. Additionally, the average survival rates of S. exigua were 52.02% and 40.38% at the pupal and adult stages, respectively, after injection with SeChi dsRNA. For the insects injected with SeChi-h dsRNA, the survival rates were 72.38% and 48.52%, respectively. These results suggest that SeChi and SeChi-h may have different biologic functions during the pupal-adult molting.

RNA interference is induced in the glassy winged sharpshooter Homalodisca vitripennis by actin dsRNA

The glassy winged sharpshooter, Homalodisca vitripennis, is an unusually robust and efficient leafhopper vector of Xylella fastidiosa. X. fastidiosa is the causative agent of Pierce's disease, almond scorch, citrus variegated chlorosis and other serious plant diseases. The present study was conducted to establish whether RNA interference (RNAi) was induced in nymphal H. vitripennis that were injected with actin dsRNAs and other dsRNAs. A dramatic reduction in target H. vitripennis actin mRNAs and the formation of small interfering RNAs (siRNAs), hallmarks of RNAi, were found following the injection of actin dsRNAs. Quantitative reverse transcription PCR indicated an 80% reduction in actin mRNA levels by 5 days post-injection. Western blot analysis showed a dramatic drop in actin protein levels by 3 days post-injection. Biological effects such as incomplete nymphal-adult ecdysis and > 95% mortality were also found following the injection of fifth-instar nymphs with actin dsRNA. Dramatic reductions in target mRNA levels were also found following the injection of other dsRNAs into fifth-instar H. vitripennis. The findings indicate that RNAi is induced in post-embryonic leafhoppers by dsRNA. The present system can be used to screen potential gene-silencing targets that can be used for reducing the vector competence of H. vitripennis and other leafhoppers.

English

Bombyxin (BBX) of the silkworm, Bombyx mori, is an insulin-like peptide (ILP). To further study the function of the BBX-B8 gene, the effects of inhibiting the expression of BBX-B8 gene on organ development, reproduction and trehalose metabolization of the silkworm were investigated. 48 h after the injection of BBX-B8 dsRNA into the 3-day larvae of fifth-instar (10 μg per larvae; 10 μl; the average weight: 3 g/larvae), the mRNA level of BBX-B8 in the brain of silkworm was declined strikingly by 37.33%; 72 h after injection, the mRNA level of BBX-B8 was decreased by 30%. After the injection of BBX-B8 dsRNA into the 3-day larvae of fifth-instar (1 or 5 μg per larvae; 10 μl), development of pupal wing imaginal discs was suppressed, more deformed adult wings were found. The number of mature eggs in the oviduct respectively increased by 7.86 and 12.62% when pupa were injected with BBX-B8 dsRNA (10 or 15 μg per larvae; 10 μl) on the first day of pupation. 72 h after the injection of BBX-B8 dsRNA (10 μg per larvae; 10 μl) into the 3-day larvae of fifth-instar, the trehalose level of hemolymph was elevated by 12.68% in silkworm. These results suggest that BBX-B8 plays an important role in organ development, reproduction and the metabolization of trehalose of silkworm.   Key words: Silkworm, insulin-like, RNAi, organ growth, reproduction.

In Vivo RNA Interference of a Gonad-Specific Transforming Growth Factor-β in the Pacific Oyster Crassostrea gigas

We investigated the role of oyster gonadal TGFβ (og-TGFβ) in the reproduction of Crassostrea gigas, using an in vivo RNA interference approach. We designed double-stranded RNA targeting og-TGFβ, which is specifically expressed in the somatic cells surrounding germ cells in the gonad of both male and female oysters. In vivo injection of this og-TGFβ dsRNA into the gonad led to knock-down phenotypes for both sexes, with significant reduction (77.52% relative to controls) of the gonad area, lowered reproductive effort and germ cell under-proliferation. Interestingly, half of the injected females halted their vitellogenesis, since we were only able to observe pre-vitellogenic oocytes. In addition, apoptotic germ cells and haemocytes infiltrated into the gonad, likely as part of the active resorption of degenerating germ cells. Conversely, males showed a normal phenotype at the cellular level, with spermatids and spermatozoids observed in the gonads of control and injected males. As a result, og-TGFβ appears to play an essential role in C. gigas germ cell development by functioning as an activator of germ cell proliferation in both male and female oysters and vitellogenesis in females.

The steroid hormone 20-hydroxyecdysone upregulated the protein phosphatase 6 for the programmed cell death in the insect midgut

Programmed cell death (PCD) plays an important role in insect midgut remodeling during metamorphosis. Insect midgut PCD is triggered by the steroid hormone 20-hydroxyecdysone (20E) and it is mediated by a series of genes. However, the mechanism by which 20E triggers midgut PCD is still unclear. Here, we report a protein phosphatase 6 (PP6) from Helicoverpa armigera playing roles in midgut PCD. PP6 was expressed in the midgut during larval growth and it is significantly increased during metamorphosis. The increase was proven to be regulated by 20E. The juvenile hormone analog methoprene has no effect on PP6 expression. RNA interference analysis suggests that 20E upregulated the PP6 transcript levels through the ecdysone receptor EcRB1. PP6 knockdown by larval feeding or PP6 dsRNA injection resulted in the repression of the midgut PCD during the metamorphic stage. The mechanism was demonstrated to be through the suppression of genes such as Broad (Br), E74a, E75b, HR3, E93, rpr, and caspase, which are involved in 20E signaling pathway or midgut PCD. These findings suggest that PP6 is involved in the 20E signal transduction pathway and participates in the PCD in midgut.

Two Major Structural Proteins Are Required for Rigid Adult Cuticle Formation in the Red Flour Beetle, Tribolium castaneum

The insect exoskeleton or cuticle is a complex extracellular matrix consisting of several functional layers. Cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo‐ and endocuticular layers or procuticle. During cuticle tanning (sclerotization and pigmentation), CPs are cross‐linked by quinones or quinone methides produced by oxidation of catechols catalyzed by the phenoloxidase, laccase. CPs contain a conserved sequence known as the Rebers & Riddiford (R&R) motif, which may function as a chitin‐binding domain that helps to coordinate the interaction between chitin fibers and the protein network. More than 100 CP‐like genes have been identified in Drosophila melanogaster, with a similar number present in the red flour beetle, Tribolium castaneum. Different CPs have been found in the different developmental stages and in different regions of the exoskeleton, indicating that specific CPs are required to produce a cuticle with the proper morphology and mechanical properties.To study the function(s) of insect CPs, we focused on adult elytra of the T. castaneum. In protein samples extracted from elytra of newly emerged adults, yielding two highly abundant proteins with apparent sizes of 10 and 20 kDa. To characterize these major proteins further, each was digested with trypsin, and the resulting peptides were analyzed by MALDI‐TOF mass spectrometry. Comparison of this result with conceptual trypsinization of the computed proteome of Tribolium identified two candidate genes, which we call TcCP10 and TcCP20. We cloned the full‐length cDNAs corresponding to these genes. Both proteins belong to the RR‐2 family (cuticle DB, http://biophysics.biol.uoa.gr/cuticleDB/) and possess a putative signal peptide sequence. TcCP20 is an ortholog of the Tenebrio adult cuticle protein, acp22. Both TcCP10 and TcCP20 were mapped to linkage group 3. The expression patterns revealed by real‐time PCR showed that the transcript levels of both genes dramatically increase at the pharate adult stage (1 ‐2 days before molting), and then decline after adult eclosion.RNA interference (RNAi) was used to investigate the functions of TcCP10 and TcCP20. Specific dsRNAs for each gene were injected into last‐instar larvae. Following dsRNA injections, expression of TcCP10 and TcCP20 genes was analyzed by real‐time PCR and SDS PAGE to evaluate transcript and protein levels. Both genes were substantially and specifically down‐regulated at the mRNA and protein levels after RNAi treatment. Injection of dsTcCP10 or dsTcCP20 had no effect on larval‐larval, larval‐pupal or pupal‐adult molts. The elytra of the resulting adults, however, were malformed and abnormal. Elytra of dsTcCP20 adults did not fully elongate and did not extend far enough to cover the abdomen. These elytra were slightly separated and not smooth as compared to those of control insects. Like the dsTcCP20 phenotype, the resulting adults obtained after injection dsTcCP10 also exhibited elytral defects, but they were more severe. These elytra were short, wrinkled, bumpy, warped and fenestrated. CP10‐deficient insects could not fold their hind wings properly and eventually died approximately 7 days after eclosion, probably because of dehydration. TcCP10 and TcCP20 were also extracted from the pronotum cuticle of Tribolium. These results demonstrate that two cuticle proteins, TcCP10 and TcCP20, play critical roles in adult cuticle formation of T. castaneum.

Expression Profiles and Functional Analysis of Genes Encoding Chitin Deacetylases, Extracellular Matrix‐Modifying Proteins in Tribolium castaneum

In insects chitin, the β‐(1,4)‐linked N‐acetylglucosamine homopolymer, is one of the major components of the exoskeleton or cuticle. The extracellular matrix (ECM) of this exoskeleton is modified in different ways to make it either very rigid and thick or thin and flexible, or to generate specialized structures such as mandibles with sharp cutting edges. Chitin deacetylases (CDAs) are secreted proteins belonging to a family of extracellular chitin‐modifying enzymes that deacetylate chitin to form chitosan, a polymer of β‐(1,4)‐linked D‐glucosamine residues. This modification possibly contributes to the affinity of chitin for a variety of proteins distinct from those that bind uniquely to chitin and also to mechanical properties of the cuticle.Two genes encoding putative chitin deacetylases in Drosophila, serpentine (CG32209) and vermiform (CG8756), play critical roles in shaping of tracheal tubes as well as in regulating the structural properties of epidermal cuticle. By searching Beetlebase (http://bioinformatics.ksu.edu/BeetleBase/), we identified nine genes encoding CDA‐like proteins in Tribolium. A comparative analysis of CDA families in other insect species including Drosophila, Anopheles and Apis indicated that the number of CDA genes varies from species to species. Of these, T. castaneum has the greatest number, including some recently duplicated genes found clustered in its genome. The expression profiles of all CDA genes during development were studied by RT‐PCR. The Tribolium genes TcCDA1 and TcCDA2 are orthologs of DmSerp and DmVerm, respectively, and are expressed throughout all stages of insect development. In contrast TcCDA6 – 9 are expressed predominantly during the larval feeding stages. in situ hybridization revealed that TcCDA1 and TcCDA2 are expressed in epidermal cells, whereas TcCDA6 through TcCDA9 are expressed in cells lining the midgut lining.To study the function(s) of TcCDA genes, double stranded RNA (dsRNA) specific for each gene was injected into insects at different developmental stages and phenotypes were observed. No visible phenotypic changes were observed after injection of dsRNAs for TcCDA3 to 9, while injection of dsRNAs for TcCDA1 or TcCDA2 affected all molts, including the larval‐larval, larval‐pupal and pupal‐adult. Insects treated with these dsRNAs did not shed their old cuticles and were trapped in the exuviae. Interestingly, different adult phenotypes were observed after injection of dsRNAs specific for alternatively spliced transcripts of TcCDA2, namely TcCDA2A and TcCDA2B. Neither dsTcCDA2A nor dsTcCDA2B had any effect on molting. However, the adults that developed after injection of dsTcCDA2A into larvae were unable to bend or unbend their legs, at the femoral‐tibial joints. In contrast, adults that emerged after larvae were injected with dsCDA2B exhibited defects in epidermal cuticle morphology, including roughened elytra. When adult females were injected with dsTcCDA1 or dsTcCDA2A, 70‐90% of their progeny were unable to hatch from the egg, and those that did hatch could not molt to second larval instar. These results support the hypotheses that; 1) TcCDA1 and TcCDA2 are critical for egg hatch and also larval‐larval, larval‐pupal and pupal‐adult molting; 2) alternatively spliced variants of TcCDA2, TcCDA2A and TcCDA2B, have different roles in determining adult cuticle morphology; and 3) Proper ratios of chitin/chitosan levels at different stages and/or in different tissues are critical for insect development.

Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: Cloning, RNAi gene silencing and protective effect of the recombinant proteins

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.

Leucomyosuppressin modulates cardiac rhythm in the cockroach Blattella germanica

Several lines of evidence point to leucomyosuppressin (LMS) and myosuppressin-related peptides as inhibitory modulators of heartbeat frequency in arthropods. Previous studies in Blattella germanica demonstrated that heartbeat frequency decreases after ootheca formation, and remains low during the period of ootheca transport. Subsequent work in this cockroach resulted in the characterization of LMS and the cloning and sequencing of its precursor. The present paper describes the activity of LMS on modulation of heartbeat in B. germanica. Assays using semi-isolated heart preparations revealed that LMS reduces heartbeat frequency in a dose dependent manner, at physiological concentrations. Additional experiments showed that LMS inhibits heartbeat rates in vivo. Finally, injection of dsRNA for LMS elicited a decrease in LMS mRNA to virtually undetectable levels and heartbeat frequency increased significantly in females carrying oothecae. These data suggest that LMS contributes to the modulation of cardiac rhythm in B. germanica during the reproductive cycle.

Large-scale RNAi screen of G protein-coupled receptors involved in larval growth, molting and metamorphosis in the red flour beetle

BackgroundThe G protein-coupled receptors (GPCRs) belong to the largest superfamily of integral cell membrane proteins and play crucial roles in physiological processes including behavior, development and reproduction. Because of their broad and diverse roles in cellular signaling, GPCRs are the therapeutic targets for many prescription drugs. However, there is no commercial pesticide targeting insect GPCRs. In this study, we employed functional genomics methods and used the red flour beetle, Tribolium castaneum, as a model system to study the physiological roles of GPCRs during the larval growth, molting and metamorphosis.ResultsA total of 111 non-sensory GPCRs were identified in the T. castaneum genome. Thirty-nine of them were not reported previously. Large-scale RNA interference (RNAi) screen was used to study the function of all these GPCRs during immature stages. Double-stranded RNA (dsRNA)-mediated knockdown in the expression of genes coding for eight GPCRs caused severe developmental arrest and ecdysis failure (with more than 90% mortality after dsRNA injection). These GPCRs include dopamine-2 like receptor (TC007490/D2R) and latrophilin receptor (TC001872/Cirl). The majority of larvae injected with TC007490/D2R dsRNA died during larval stage prior to entering pupal stage, suggesting that this GPCR is essential for larval growth and development.ConclusionsThe results from our study revealed the physiological roles of some GPCRs in T. castaneum. These findings could help in development of novel pesticides targeting these GPCRs.

Contribution of Ldace1 gene to acetylcholinesterase activity in Colorado potato beetle

The Colorado potato beetle (CPB), Leptinotarsa decemlineata is an important economic pest of potato worldwide. Resistance to organophosphates and carbamates in CPB has been associated in some cases to point mutations in the acetylcholinesterase (AChE) gene Ldace2, an orthologue of Drosophila melanogaster Dmace2. In this paper we report cloning and sequencing of Ldace1, an orthologue of Anopheles gambiae Agace1 that was previously unknown in CPB. The Ldace1 coding enzyme contains all residues conserved in a functionally active AChE. Ldace1 is expressed at higher levels (between 2- and 11-fold) than Ldace2 in embryos, in the four larval instars and in adults. Specific interference of Ldace1 by means of dsRNA injection resulted in a reduction of AChE activity to an approximate 50% compared to control, whilst interference of Ldace2 reduced AChE activity to an approximate 85%. Analysis of zymograms of AChE activity after interference indicates that LdAChE1 is the enzyme predominantly responsible for the activity visualised. Interference of Ldace1 in CPB adults caused a significant increase in mortality (43%) as early as three days post-injection (p.i.), suggesting the essential role of Ldace1. Interference of Ldace2 also caused a significant increase in mortality (29%) compared to control, although at seven days p.i. The effect of the interference of Ldace1 on susceptibility to the organophosphate chlorpyrifos points out that LdAChE1 could be a main target for this insecticide. In the light of our results, studies associating resistance in CPB to mutations in Ldace2 should be reviewed, taking into consideration analysis of the Ldace1 gene.

RNAi knock‐downs support roles for the mucin‐like (AeIMUC1) gene and short‐chain dehydrogenase/reductase (SDR) gene in Aedes aegypti susceptibility to Plasmodium gallinaceum

The mosquito midgut represents the first barrier encountered by the Plasmodium parasite (Haemosporida: Plasmodiidae) when it is ingested in blood from an infected vertebrate. Previous studies identified the Aedes aegypti (L.) (Diptera: Culicidae) mucin-like (AeIMUC1) and short-chain dehydrogenase/reductase (SDR) genes as midgut-expressed candidate genes influencing susceptibility to infection by Plasmodium gallinaceum (Brumpt). We used RNA inference (RNAi) by double-stranded RNA (dsRNA) injections to examine ookinete survival to the oocyst stage following individual gene knock-downs. Double-stranded RNA gene knock-downs were performed 3 days prior to P. gallinaceum infection and oocyst development was evaluated at 7 days post-infection. Mean numbers of parasites developing to the oocyst stage were significantly reduced by 52.3% in dsAeIMUC1-injected females and by 36.5% in dsSDR-injected females compared with females injected with a dsβ-gal control. The prevalence of infection was significantly reduced in dsAeIMUC1- and dsSDR-injected females compared with females injected with dsβ-gal; these reductions resulted in a two- and three-fold increase in the number of uninfected individuals, respectively. Overall, these results suggest that both AeIMUC1 and SDR play a role in Ae. aegypti vector competence to P. gallinaceum.

Penaeus monodon is protected against gill- associated virus by muscle injection but not oral delivery of bacterially expressed dsRNAs

Gill-associated virus (GAV) is a nidovirus that commonly infects Penaeus monodon (black tiger shrimp) in eastern Australia, causing morbidity and mortalities in the acute stage of disease. Here we explored the possibility of inhibiting GAV replication and disease using double-stranded (ds)RNAs expressed in bacteria and delivered either orally or by muscle injection. To enhance potential RNA interference (RNAi) responses, 5 long dsRNAs were used that targeted open reading frame 1a/1b (ORF1a/b) gene regions and thus only the genomic length RNA. To examine oral delivery, P. monodon were fed pellets incorporating a pool of formalin-fixed bacteria containing the 5 GAV-specific dsRNAs before being injected with a minimal lethal GAV dose. Feeding with the pellets continued post-challenge but did not reduce mortality accumulation and elevation in GAV loads. In contrast, muscle injection of the dsRNAs purified from bacteria was highly effective at slowing GAV replication and protecting shrimp against acute disease and mortalities. In synergy with these data, dsRNA targeted to P. monodon beta-actin mRNA caused 100% mortality following injection, whilst its oral delivery caused no mortality. Findings confirm that injected dsRNA can mount effective RNAi responses in P. monodon to endogenous shrimp mRNA and exogenous viral RNAs, but when delivered orally in bacteria as a feed component, the same dsRNAs are ineffective. The efficacy of the RNAi response against GAV provided by injection of dsRNAs targeted to multiple genome sites suggests that this strategy might have general applicability in enhancing protection against other shrimp single-stranded (ss)RNA viruses, particularly in hatcheries or breeding programs where injection-based delivery systems are practical.

RNAi Interference by dsRNA Injection into <em>Drosophila</em> Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process (1). Over the years many improvements and tools for genetic manipulation have become available in Drosophila (2). Soon after the initial discovery by Frie and Mello (3) that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development (4, 5). Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases (6, 7). The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs (8). The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion (9, 10). dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes required for tissue and organ development in Drosophila.

RNAi Interference by dsRNA Injection into <em>Drosophila</em> Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5. Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.