A baculovirus enhancin alters the permeability of a mucosal midgut peritrophic matrix from lepidopteran larvae

The peritrophic matrix (PM) in lepidopterous larvae may function as a defensive barrier against ingested viral pathogens. PMs isolated from Trichoplusia ni and Pseudaletia unipuncta larvae, were treated with a baculovirus-encoded metalloprotease (enhancin) from Trichoplusia ni granulosis virus (TnGV) and their in vitro permeability to blue dextran and fluorescent-labelled Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined using a dual chamber permeability apparatus. Incubation of T. ni PMs with 0.0, 0.5, 1.0, and 2.0mg/ml enhancin resulted in a blue dextran 2000 flux of 4.4, 6.3, 9.9, and 15.6&mgr;g/mm(2)/h, respectively. In addition, T. ni PMs treated with enhancin were found to be significantly more permeable to fluorescent-labelled AcMNPV than non-treated control PMs. The permeability of T. ni PMs treated with 3.0mg/ml enhancin was 0.017 cumulative percent crossing/mm(2)/h, whereas the permeability of the control PM was below the detectable limit. Similarly, enhancin treatment greatly increased the permeability of P. unipuncta PMs to AcMNPV. These results provide evidence that the PM from two lepidopteran species can block the passage of baculovirions across this matrix thus reducing the probability of larval infection. Furthermore, these results support the hypothesis that enhancin facilitates NPV infection of larvae by altering the permeability of the PM.     

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.     

杆状病毒AcMNPV p35基因的克隆表达及多抗制备

用PCR方法扩增得到苜蓿丫纹夜蛾核多角体病毒(Autographa californic anucleopolyhedrovirus,AcM N-PV)p35基因,将其克隆至质粒pET-32a( )上,构建得到重组质粒pET-p35,转化大肠杆菌BL21(DE3),经IPTG诱导,表达了1条约为55 ku的蛋白带。以Ni2 -NTA偶连抗体检测证明所表达的蛋白为带有组氨酸的融合蛋白。采用割胶回收的方法纯化融合蛋白,以纯化的融合蛋白制备多克隆抗体,效价为1/1 024。免疫印迹分析表明,该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。     

Effect of plant patch shape on the distribution and abundance of three lepidopteran species associated with Brassica oleracea

• 1 Plant patch shape may affect the abundance of herbivorous insects. Patches of the same size but longer or irregular have a higher perimeter/area relationship (P/A) than square or regular ones, which may determine the immigration, emigration and abundance of individuals in the patch.
• 2 Only specialist species should be affected by plant patch shape. Those species that are more abundant in smaller patches should be more abundant in patches with higher P/A, whereas those that are more abundant in larger patches should be more abundant in patches with lower P/A.
• 3 We studied the density of eggs, larvae and pupae of Pieris brassicae, Plutella xylostella and Trichoplusia ni in square (low P/A) and I‐shaped (high P/A) patches of 144 plants of Brassica oleracea. We also estimated their immigration to these patches, and the final plant weight.
• 4 Plant patch shape affected the abundance, but not the distribution, of the two specialist species. Whereas P. brassicae was denser in I‐shaped patches, P. xylostella was more abundant in square patches. The generalist T. ni was not affected by patch shape. Immigration of P. brassicae was higher in I‐shaped patches, but immigration of P. xylostella and T. ni was not affected by patch shape. Plants were heavier in the centre of square patches.
• 5 Our results suggest that plant patch shape affects the density of herbivorous insects and should be considered independently from other plant patch variables when studying the population dynamics of these organisms.

     

Serial Selection for Resistance to a Wild-Type and to a Genetically Modified Nucleopolyhedrovirus in Trichoplusia ni

In previous work, cabbage loopers (Trichoplusia ni) evolved 22-fold resistance to the single nucleocapsid nucleopolyhedrovirus of T. ni (TnSNPV) after 26 generations of selection with the virus. The goal of the present study was to determine if T. ni could evolve resistance to the recombinant Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV-AaIT) that expresses an insect specific neurotoxin and to determine if it was influenced by prior development of resistance to TnSNPV. To answer these questions, the T. ni line that had been exposed to TnSNPV was divided into two sublines at generation 27. One of them was serially selected for resistance to AcMNPV-AaIT (subline TnSNPV/AcMNPV-AaIT), while the other one was mock infected with distilled water (subline TnSNPV/H₂O). The same was done with the line that was used as a control from generations 1 to 26 (subline H₂O/AcMNPV-AaIT and subline H₂O/H₂O). After 17 generations of selection with AcMNPV-AaIT, T. ni that had not been previously exposed to TnSNPV evolved only twofold resistance to AcMNPV-AaIT. However, those that had been selected with TnSNPV evolved fourfold resistance to AcMNPV-AaIT. Exposure to AcMNPV-AaIT conferred cross-resistance to TnSNPV in only one subline, subline H₂O/AcMNPV-AaIT. Resistance to AcMNPV-AaIT did not affect the developmental time, pupal weight, egg production, or percentage of egg hatch of T. ni.     

The role of viral protein Ac34 in nuclear relocation of subunits of the actin-related protein 2/3 complex

The actin nucleator actin-related protein complex(Arp2/3) is composed of seven subunits: Arp2,Arp3, p40/ARPC1(P40), p34/ARPC2(P34), p21/ARPC3(P21), p20/ARPC4(P20), and p16/ARPC5(P16). Arp2/3 plays crucial roles in a variety of cellular activities through regulation of actin polymerization. Autographa californica multiple nucleopolyhedrovirus(Ac MNPV), one of the beststudied alphabaculoviruses, induces Arp2/3 nuclear relocation and mediates nuclear actin polymerization to assist in virus replication. We have demonstrated that Ac34, a viral late-gene product, induces translocation of the P40 subunit of Arp2/3 to the nucleus during Ac MNPV infection. However, it remains unknown whether Ac34 could relocate other Arp2/3 subunits to the nucleus. In this study, the effects of the viral protein Ac34 on the distribution of these subunits were studied by an immunofluorescence assay. Arp2, P34, P21, and P20 cloned from Spodoptera frugiperda(Sf9) cells showed mainly cytoplasmic localization and were relocated to the nucleus in the presence of Ac34. In addition, Arp3 was localized in the cytoplasm in both the presence and absence of Ac34, and P16 showed whole-cell localization. In contrast to Sf9 cells, all subunits of mammalian Arp2/3 showed no nuclear relocation in the presence of Ac34. Co-immunoprecipitation analysis of the interaction between Ac34 and Arp2/3 subunits revealed that Ac34 bound to P40,P34, and P20 of Sf9 cells. However, none of the subunits of mammalian Arp2/3 interacted with Ac34, indicating that protein-protein interaction is essential for Ac34 to relocate Arp2/3 subunits to the nucleus.     

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.     

Systematic Analysis of 42 Autographa Californica Multiple Nucleopolyhedrovirus Genes Identifies An Additional Six Genes Involved in the Production of Infectious Budded Virus

Baculoviruses have been widely used as a vector for expressing foreign genes. Among numerous baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most frequently used and it encodes 155 open reading frames (ORFs). Here, we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses (BVs) by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays. The results showed that among the 39 functionally unverified genes and 3 recently reported genes, 36 are dispensable for infectious BV production, as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus. Three genes (ac62, ac82 and ac106/107) are essential for infectious BV production, as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus. In addition, three genes (ac13, ac51 and ac120) are important but not essential for infectious BV production, as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses. We then grouped the 155 AcMNPV genes into three categories (Dispensable, Essential, or Important for infectious BV production). Based on our results and previous publications, we constructed a schematic diagram of a potential mini-genome of AcMNPV, which contains only essential and important genes. The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.     

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.     

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: Large scale production of the E1 protein

The two envelope glycoproteins of rubella virus (RV), El of 58 kDa and E2 of 42–47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10−1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.     

Developmental Changes in the Structure of Endosperm Starch of Rice (Oryza sativa L.)

The developmental changes in the structure and properties of endosperm starches were investigated using the near-isogenic lines for wx alleles of rice. The amylose content in nonwaxy starch was increased during the development of rice grains. Because the accumulation of amylose in endosperm stopped earlier than that of amylopectin during development, the percentages of amylose reached a maximum at the 17th day after flowering in nonwaxy endosperm. Since the distributions of the unit-chain length of amylopectin in waxy and nonwaxy starches were unchanged with the development of the grains, these amylopectins would be synthesized in a similar manner through development. The structure and properties of endosperm starches were reconfirmed to be conspicuously affected by the temperature at the early developmental stages of the grain-filling period, namely, they appeared to be characterized by the temperature at which the starch was accumulated in the endosperm.     

The role of peritrophic membrane in the resistance of Anticarsia gemmatalis larvae (Lepidoptera: Noctuidae) during the infection by its nucleopolyhedrovirus (AgMNPV)

The aim of this study was to analyze morphologically the peritrophic membrane (PM) of Anticarsia gemmatalis larvae resistant (RL) and non-resistant (susceptible) (SL) to the A. gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV), in the presence of viral infection. Also, in this investigation the results between SL and RL were compared to improve the understanding of the resistance mechanisms to the virus. The PM of SL of A. gemmatalis was less efficient as a barrier against the viral infection since it was found to be more fragile than the PM of RL. The lower chitin content as seen from weaker fluorescent staining in SL as well as the abundance of non-solubilized vesicular materials in the ectoperitrophic space, would cause the malformation of this membrane, facilitating the passage of the virus toward the epithelium of the midgut. On the other hand, in RL, the intensity of WGA (wheat germ agglutinin)-conjugated FITC (fluorescein) reaction of the PM was greater than in SL, making this insect more resistant to infection. We can conclude that the effectiveness of the PM in protecting against pathogens is dependent on the integrity of the epithelial cells of the midgut and of the structural preservation of the PM, being directly implicated in the resistance of A. gemmatalis larvae to AgMNPV.     

Circadian changes in melatonin in the nervous system and hemolymph of the cabbage looper moth,Trichoplusia ni

Quantitative levels of melatonin and 5-hydroxytryptamine were measured over the scotophase in the protocerebrum, subesophageal ganglion, optic lobes, thoracic ganglia, and hemolymph of adult male cabbage looper moths, Trichoplusia ni, using HPLC with electrochemical detection. Melatonin levels were very low (s < 1 pmol) or undetectable during the photophase, but increased in all tissues during the dark. Lowest mean levels in the dark were observed in the optic lobes (0.3 to 0.7 pmols). Maximal mean levels in the protocerebrum (5.2 pmols) occurred in the early part of the scotophase, but in all other tissues (2.8 in the subesophageal ganglion; 9.5 in thoracic ganglia) and hemolymph (18 pg/l) maximal mean levels were observed later in the dark. Levels of 5-hydroxytryptamine in each tissue, in contrast, were higher than melatonin levels in the photophase, and in the protocerebrum and thoracic ganglia decreased during the dark, but in the optic lobes and subesophageal ganglion remained unchanged. Further, decreases in 5-hydroxytryptamine during the dark were significantly lower than the increased levels of melatonin, suggesting that active synthesis of 5-hydroxytryptamine was occurring. In a second experiment, when measured from individuals in three different photoperiods (618, 1212, 186 lightdark) maximum mean melatonin levels in the brain (protocerebral and subesophageal ganglia) peaked within the first 1.5 h of the dark and remained at measurable levels for the duration of the dark. In a third experiment, levels of melatonin in the brain and thoracic ganglia displayed rhythmicity in continuous dark conditions but not in continuous light, when compared with profiles obtained in a normal light dark regime.Abbreviations CNS Central nervous system - HPLC-ECD high performance liquid chromatography with electrochemical detection - MEL melatonin - 5HT 5-hydroxytryptamine - N-ac-SHT N-acetyl-5-hydroxytryptamine     

Effects of crop type on Bacillus thuringiensis toxicity and residual activity against Trichoplusia ni in greenhouses

We assessed the efficacy and persistence of a Bacillus thuringiensiskurstaki (Btk) formulation (Dipel) against Trichoplusia ni (Hubner) (Lep., Noctuidae), the cabbage looper, on three greenhouse vegetable crops (tomato, bell pepper and cucumber). First, T. ni larvae were fed leaf discs treated with Btk to assess how Btk toxicity varies with host plant. Secondly, T. ni larvae were fed leaf discs harvested from plants that had been sprayed with Btk 1, 5 and 9 days previously to assess the residual activity of Btk toxicity in greenhouse environments. Mortality of T. ni larvae fed tomato leaf discs was significantly higher than T. ni fed cucumber or pepper leaf discs. The toxicity of Btk had declined by less than 50% after 9 days, which suggests that Btk persistence is lengthy in greenhouse environments. No crop effects on the residual activity of Btk were found. These results demonstrate that the greenhouse environment and the crop should be considered when using Btk for insect management on greenhouse crops.     

Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae

The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine-rich polydnavirus genes have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine-rich Vhv1.4 protein. Full- length and truncated Vhv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the Vhv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The Vhv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The Vhv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the Vhv1.4 protein is glycosylated at multiple N-glycosylation sites. Immunofluorescence assays showed that the Vhv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the Vhv1.4 protein was internalized, probably by endocytosis. Specific binding of the Vhv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response. Arch. Insect Biochem. Physiol. 36:251–271, 1997. © 1997 Wiley-Liss, Inc.     

Effects of infection with a granulosis virus on larval growth, development and ecdysteroid production in the cabbage looper, Trichoplusia ni

ABSTRACT. Granulosis virus-infected Trichoplusia ni (Hûbner) larvae exhibited an increased larval life span with no supernumerary moult and no pupation. Weight gain was not affected. Insects infected shortly after hatching were slower in reaching the fourth and fifth stadia than were control insects. Haemolymph ecdysteroid titres were lower in virus-infected insects than control insects, but these differences were only significant ( P <0.05) in the fifth stadium. Electron microscopic examination of the pro thoracic glands revealed extensive granulosis virus infection, and glands from virus-infected insects produced no RIA-detectable ecdysteroids in vitro. Injection of 20-OII-ecdysone into virus-infected larvae at various concentrations and times did not induce pupation.     

Effect of chemical complexity of essential oils on feeding deterrence in larvae of the cabbage looper

The relationship between chemical composition and feeding deterrent activity of seven essential oils is explored, including those from Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae), Cinnamomum zeylanicum Blume (Lauraceae), Lavendula latifolia (L.) Medicus, Lavendula angustifolia L., Mentha crispa L., Mentha arvensis L. and Mentha piperita L. (Lamiaceae) against an important agricultural pest, the cabbage looper (Trichoplusia ni Hübner, Noctuidae), using a leaf‐disc choice bioassay. Comparison of the deterrent activity of ‘full mixtures' with respective artificial blends missing individual constituents demonstrates that, for most oils, minor constituents in a mixture can be as important as major constituents with respect to the overall feeding deterrent effect. There is a lack of correlation between the feeding deterrent effect of an individual constituent of an essential oil and its contribution to the overall activity of the ‘full mixture’ in some cases. The effect of removing an individual constituent from the mixture of an essential oil depends on the unique properties of the mixture, which in turn may reflect the interaction of its constituents. Understanding the role and contribution of each constituent to the overall activity of the oil can facilitate the creation of artificial blends that optimize their efficacy against different pests.