Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.     

Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris)

ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.     

烟芽夜蛾囊泡病毒3h株编码的3H-117蛋白能干扰AcMNPV的口服活性

[目的]囊泡病毒是一类体液传播的昆虫病毒,具有特殊的致病历程和良好的生防应用潜力.烟芽夜蛾囊泡病毒3h株(Heliothis virescens ascovirus 3h,HvAV-3h)是我国分离的第一株囊泡病毒,其编码的3H-117蛋白被报道为HvAV-3h病毒粒子的结构蛋白.[方法]为了进一步探究3h-117基因...     

Baculovirus-mediated Expression of a Gene for Trehalase of the Mealworm Beetle, Tenebrio molitor, in Insect Cells, SF-9, and Larvae of the Cabbage Armyworm, Mamestra brassicae

It is of interest to understand what kinds of physiological and biochemical changes occur in insects if the homeostasis of trehalose in the hemolymph is disrupted by the infection with a recombinant baculovirus containing a secretory-trehalase gene. For this purpose, two recombinant non-occluded Autographa california multicapsid nucleopolyhedroviruses (AcMNPVs), vTREVL and vERTVL, containing a trehalase cDNA of the mealworm beetle, Tenebrio molitor, were constructed. The trehalase cDNA was inserted in the sense orientation downstream of the polyhedrin promoter for vTREVL, and in the anitsense orientation for vERTVL. The active trehelase of T. molitor was found outside of cells when SF-9 cells or larvae of the cabbage armyworm, Mamestra brassicae, were infected with vTREVL. In the hemolymph of vTREVL-infected larvae, expression of the active trehelase was followed by disappearance of trehalose and appearance of glucose. However, the mortality time of virus-infected 5th instar larvae increased in the following order: AcMNPV C6 (wild-type virus) ≤ vERTVL < vTREVL. The symptoms (the browning and liquefying of the host body) of NPV infection were moderated considerably in vTREVL-infected larvae.     

Greenhouse Evaluation of Dose– and Time–Mortality Relationships of Two Nucleopolyhedroviruses for the Control of Beet Armyworm, Spodoptera exigua, on Chrysanthemum

Dose– and time–mortality relationships of baculoviruses in pest insects are important for the determination of effective spraying regimes. A series of experiments with Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV) against synchronized populations of S. exigua larvae in greenhouse chrysanthemum was conducted. Dose– and time–mortality relationships of different virus concentrations and S. exigua target stages were determined and the area foliage consumption was measured. Crop injury was greatly reduced when S. exigua were controlled as second or third instar larvae, whereas virus applications against fourth instar larvae could not prevent considerable crop injury, even at high concentrations. SeMNPV was approximately 10 times as infectious as AcMNPV when applied on greenhouse chrysanthemum. The relative virulence of AcMNPV and SeMNPV corresponded reasonably well with previously published laboratory bioassay data. SeMNPV killed second and fourth instar S. exigua larvae approximately 12 h faster than did AcMNPV in chrysanthemum, but no difference in speed of action was found for third instar larvae. The relative speed of action of AcMNPV and SeMNPV determined in chrysanthemum and in laboratory bioassays did not correspond for third instar S. exigua larvae; laboratory bioassay data can therefore not simply be extrapolated to the crop level.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

缺失宿主Vta1蛋白的MIT结构域对杆状病毒AcMNPV复制的影响

【目的】克隆草地贪夜蛾(Spodoptera frugiperda)Vta1基因,检测Vta1在苜蓿银纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)复制中的作用。【方法】利用反转录-PCR与PCR方法筛选草地贪夜蛾Vta1基因及缺失Vta1N-端MIT结构域的突变体并构建其瞬时表达质粒,通过转染Sf9细胞检测表达;构建Vta1及其突变体的双分子荧光互补表达质粒,并通过瞬时转染检测其与Vps4及ESCRT-III亚基Vps46与Vps60的相互作用;共转染gp64与Vta1及其突变体瞬时表达质粒,检测瞬时表达Vta1突变体对AcMNPV出芽型病毒产量及病毒基因启动子指导报告基因表达的影响。【结果】获得了草地贪夜蛾Vta1基因。氨基酸序列相似性分析表明,昆虫、酵母与人类Vta1同源蛋白的相似性分别约为20%与50%。Western blotting分析表明GFP标签的Vta1及其突变体均能在瞬时转染的Sf9细胞中表达。双分子荧光互补分析发现,缺失第1个或第2个MIT结构域显著降低Vta1突变体与Vps4、Vps46或Vps60的相互作用。此外,瞬时表达Vta1突变体显著降低了AcMNPV感染性出芽型病毒的产量,但并未影响AcMNPVie1基因早期启动子和p6.9基因晚期启动子指导的LacZ和GUS报告基因的表达。【结论】Vta1可能参与杆状病毒AcMNPV子代病毒粒子的组装和/或出芽释放过程。     

NeuroBactrus,a Novel,Highly Effective,and Environmentally Friendly Recombinant Baculovirus Insecticide

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin–cry1-5–polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin–Cry1-5–polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.     

Characterization of a Variant ofAutographa californicaNuclear Polyhedrosis Virus with a Nonfunctional ORF 603

A new genotypic variant ofAutographa californicanuclear polyhedrosis virus (AcMNPV), the V8 variant, was originally identified by an additionalHindIII site in theHindIII–F fragment. Insect bioassays of this variant displayed a decreased time of mortality compared with the L1 variant of AcMNPV inSpodoptera frugiperdalarvae but not inTrichoplusia nilarvae. A 1.8-kb region containing the 3′ end of ORF 5,lef-2,ORF 603, and the 5′ end of the polyhedrin gene (polh) of both L1 and V8 was sequenced. V8 exhibited extensive sequence variation in the region between the 3′ end oflef-2and the 5′ end ofpolh; V8 had six amino acid substitutions in thelef-2gene product and a nonfunctional ORF 603. A site-specific frameshift mutation in ORF 603 of the L1 variant was constructed to determine the effect of ORF 603 inS. frugiperdalarvae. Truncation of ORF 603 was found to decrease the time of mortality inS. frugiperdalarvae. The insect-selective toxin gene,tox34, was inserted into the V8 variant by direct cloning. The efficacy of this recombinant as a biopesticide was equivalent to similar L1 recombinants.     

Effect of dietary selenium supplementation on resistance to baculovirus infection

We have examined the effects of dietary selenium (Se) supplementation on larval growth and immunocompetence of the lepidopteran pest, the cabbage looper, Trichoplusia ni. Supplementation of the diet of T. ni larvae with 10–20 ppm Se resulted in a 1 day delay in pupation. The effects of the addition and/or removal of dietary Se on total Se bioaccumulation and sequestration were determined by neutron activation analysis of pupae. Early penultimate instar larvae moved from selenium containing diet to basal diet lost total pupal Se content down to the level of those fed basal diet. Conversely, larvae moved from basal diet to diet containing additional Se rapidly attained pupal Se levels comparable to larvae fed Se throughout larval development. Therefore, dietary Se is rapidly accumulated or lost during larval development, but significant amounts are sequestered from diet into pupae. Larvae were reared on diet supplemented with 5 or 10 ppm Se until the onset of the penultimate instar then infected per os with increasing concentrations of the fatal baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Larvae fed Se in the penultimate and ultimate instars were more resistant to viral infection than larvae not fed Se in the final instars. This study indicates that dietary Se levels rapidly impact Se assimilation and sequestration and that tissue Se levels are an important factor in resistance to AcMNPV infection in larval T. ni.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

Handling time, prey preference, and functional response for Chrysoperla rufilabris in the laboratory

The predaceous larvae of Chrysoperla rufilabris (Burmeister) exhibited some success-motivated searching, particularly when feeding on Heliothis virescens (F.) eggs, but handling time did not decrease with experience. Handling time for H. virescens larvae was more than twice that for eggs. H. virescens larvae were preferred to cotton aphids (Aphis gossypii (F.)) while aphids were preferred to H. virescens eggs. C. rufilabris larvae exhibited a linear functional response to the three prey types tested, over the prey densities tested.USDA, ARS, Retire. Present address: 200 Highland, College Station, TX 77840, USA     

Impact of Recombinant Baculovirus Applications on Target Heliothines and Nontarget Predators in Cotton

Recombinant baculoviruses have been genetically engineered to reduce the time to kill infected pests, thus reducing crop damage. In this study, wild-type viruses and recombinant viruses expressing a scorpion toxin were applied to cotton in response to larval infestations of Helicoverpa zea and Heliothis virescens in 1997 and 1998. A chemical standard and an untreated control acted as comparison treatments. The goals of this field study were to (1) assess the efficacy of recombinant baculoviruses in protecting cotton from larval feeding damage; (2) assess the impact of recombinant virus introductions on predator densities and diversity; and (3) determine if cotton predators acquire baculovirus by consuming infected heliothines. When applications were timed at larval emergence, certain recombinant virus treatments protected cotton from damage better than wild-type virus treatments and as well as the chemical standard. Differences in efficacy between recombinant and wild-type baculoviruses were not apparent if treatments were applied 3 to 4 days after peak larval emergence. Predator densities and diversity were similar among recombinant and wild-type baculovirus treatments, whereas plots treated with the chemical standard had consistently smaller predator populations. From polymerase chain reaction analyses of predators in 1997 and 1998, 1.7 and 0.2%, respectively, of predators had consumed a virus-infected heliothine. Nine of the 26 predators carrying viral DNA were positive for recombinant virus. Additionally, 13 of the 26 predators were found to disperse 13.5 to 105 m 2 to 5 days after initial virus applications. Five of these dispersing predators (0.2% of all predators evaluated) carried recombinant viral DNA. These results suggest that the potential for the inadvertent spread of recombinant viral DNA via dispersing predators is low.     

Aerosol infectivity of a Baculovirus to Trichoplusia ni larvae: An alternative larval inoculation strategy for recombinant protein production

The baculovirus–insect expression system is a popular tool for recombinant protein production. The standard method for infecting insect larvae with recombinant baculovirus for protein production involves either feeding occlusion bodies or injecting budded virus into the cuticle. In this study, we showed that the recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at titers >10⁸ pfu/mL efficiently infected Trichoplusia ni (T. ni) larvae through aerosol inoculation of budded virus at a pressure of 5.5 × 10⁴ Pa. The dipping T. ni larvae in virus‐containing solution efficiently infected them. These results indicate that surface contamination, either by aerosol or dipping, lead to infection via spiracles. The aerosol infection route for AcMNPV was restricted to T. ni and Plutella xylostella larvae, whereas Spodoptera litura and Helicoverpa armigera larvae were resistant to this inoculation process. The yields of the reporter proteins DsRed and EGFP from T. ni larvae following aerosol infection were nearly identical to those following oral feeding or injection. This alternative baculovirus infection strategy facilitates recombinant protein and virus production by insect larvae. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cry2A toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> expressed in insect cells are toxic to two lepidopteran insects

The cry2Aa and cry2Ab genes from a Brazilian Bacillus thuringiensis strain were introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate the heterologous proteins expression in insect cells and their toxicity to different insects. The recombinant viruses (vAcCry2Aa and vSynCry2Ab) were amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analysed by SDS-PAGE, which detected the presence of polypeptides around 65 kDa. Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using the heterologous proteins showed toxicity against second instar A. gemmatalis larvae (Cry2Aa) with a LC₅₀ of 1.03 μg/ml and second instar S. frugiperda larvae (Cry2Ab) with a LC₅₀ of 3.45 μg/ml. No toxic activity was detected for Aedes aegypti and Culex quinquenfaciatus.     

Inapparent infection ofDiatraea grandiosella byHeliothis virescens cytoplasmic polyhedrosis virus

The terminal stage of infection with cytoplasmic polyhedrosis viruses (CPVs) is formation of crystal-like inclusion bodies (polyhedra) in host insects. The degree of susceptibility of larvae to CPV, based on light microscopy and presence of polyhedra, varies with the host species.Heliothis virescens (F.) andSpodoptera exigua (Hübner) are highly susceptible to CPV. In CPV treatedDiatraea grandiosella (Dyar), polyhedra were absent in all 400 + insects examined with light and electron microscopy. However,H. virescens larvae became infected when fed haemolymph ofD. grandiosella larvae or pupae (36±10 days post treatment) developed from CPV-treated larvae. No difference in pathology was observed betweenH. virescens larvae infected with CPV polyhedra and haemolymph fromD. grandiosella. This study provides evidence thatD. grandiosella can serve as a symptomless (no occlusion bodies) carrier of a CPV which is fully expressed inH. virescens species. The observation is interesting because it reveals a potentially important aspect of the epizootiology of this insect virus.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

A novel recombinant baculovirus expressing insect neurotoxin and producing occlusion bodies that contain Bacillus thuringiensis Cry toxin

A novel recombinant baculovirus, designated AcB5A, was constructed to develop an improved baculovirus insecticide with additional beneficial properties. Bacillus thuringiensis crystal protein gene (cry1–5) and an insect-specific neurotoxin gene (AaIT) were introduced into the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin–cry1–5–polyhedrin under the control of polyhedrin gene promoter, and by insertion of AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus. RT-PCR analysis with total RNA from AcB5A-infected cells indicated that cry1–5 and AaIT genes were normally transcribed. The 150 kDa of polyhedrin–Cry1–5–polyhedrin fusion protein was produced by AcB5A and occluded into polyhedra produced by the recombinant virus. This protein was activated when treated with trypsin to form a crystal protein of approximately 65 kDa. The AcB5A showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the lethal time against Spodoptera exigua larvae compared to those infected with wild-type AcMNPV. The expression level of the fusion protein decreased after in vivo passage as a result of homologous recombination between the two polyhedrin genes.