Chie Goto

Re-Evaluation of the PBAN Receptor Molecule: Characterization of PBANR Variants Expressed in the Pheromone Glands of Moths.

Sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR) activation. PBANR was initially cloned from pheromone glands (PGs) of Helicoverpa zea and Bombyx mori. The B. mori PBANR is characterized by a relatively long C-terminus that is essential for ligand-induced internalization, whereas the H. zea PBANR has a shorter C-terminus that lacks features present in the B. mori PBANR critical for internalization. Multiple PBANRs have been reported to be concurrently expressed in the larval CNS of Heliothis virescens. In the current study, we sought to examine the prevalence of multiple PBANRs in the PGs of three moths and to ascertain their potential functional relevance. Multiple PBANR variants (As, A, B, and C) were cloned from the PGs of all species examined with PBANR-C the most highly expressed. Alternative splicing of the C-terminal coding sequence of the PBAN gene gives rise to the variants, which are distinguishable only by the length and composition of their respective C-terminal tails. Transient expression of fluorescent PBANR chimeras in insect cells revealed that PBANR-B and PBANR-C localized exclusively to the cell surface while PBANR-As and PBANR-A exhibited varying degrees of cytosolic localization. Similarly, only the PBANR-B and PBANR-C variants underwent ligand-induced internalization. Taken together, our results suggest that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species. The high GC content of the C-terminal coding sequence in the B and C variants, which makes amplification using conventional polymerases difficult, likely accounts for previous “preferential” amplification of PBANR-A like receptors from other species.

Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel Bombyx mori Nucleopolyhedrovirus-Based Vector with a Modified fp25K Gene

ABSTRACT Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

In vivo characterization of two granuloviruses in larvae of Mythimna separata (Lepidoptera: Noctuidae)

The pathogenicity of two granuloviruses (GVs), Xestia c-nigrum GV (XecnGV) and Pseudaletia unipuncta GV (PsunGV), was examined in Mythimna separata. Partial sequencing of the genome of PsunGV indicated that it is related closely to XecnGV, but considered to be a different species. PsunGV and XecnGV showed similar pathogenicity in terms of dose-mortality response and pattern of host mass changes following infection. Both GVs killed infected larvae in 2-3 weeks. Temporal changes in the concentrations of GV-specific DNA in the larval haemolymph were measured by using a real-time quantitative PCR. Viral DNA concentration increased quickly and reached a plateau at 60-72 h post-inoculation. Rates of budded virus (BV) production of each GV were estimated on the basis of viral DNA concentrations by a modified Gompertz model. The slopes of the estimated BV growth curves of both XecnGV and PsunGV in M. separata larvae were equivalent to that of Mamestra brassicae nucleopolyhedrovirus (NPV) in its original host, reported in our previous study. This suggested that BV production is not a major factor in the slower killing speed of GVs in comparison to NPVs. The GV-infected larvae survived for an additional 10 days or more after reaching a maximum level of BV concentration, and kept growing without pupation. These findings also suggested that the GVs have a unique mechanism to regulate the growth of host larvae.

Biological activity and identification of nucleopolyhedroviruses isolated from Mythimna separata and Spodoptera litura in Japan

We compared the infectivity of two nucleopolyhedroviruses (NPVs), MyseNPV G isolated from Mythimna separata (Walker) (Lepidoptera: Noctuidae) and SpltNPV S isolated from Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). MyseNPV G was more pathogenic against M. separata than against S. litura. Although SpltNPV S was more pathogenic than MyseNPV G against S. litura, it did not infect M. separata. Restriction endonuclease (REN) analysis of viral genomic DNA revealed that the two NPVs have quite different REN profiles. Based on nucleotide sequences of the coding regions of polyhedrin, lef-8 and lef-9, SpltNPV S was closely related to other SpltNPV isolates, whereas MyseNPV G appeared to belong to the Mamestra NPV group, and was distinct from a Chinese isolate of Leucania (=Mythimna) separata NPV. The potential of MyseNPV G and SpltNPV S to control pest insects is discussed.

Enhancement of nucleopolyhedrovirus infectivity against Mamestra brassicae (Lepidoptera : Noctuidae) by proteins derived from granulovirus and a fluorescent brightener

Abstract The synergistic enhancement of nucleopolyhedrovirus (NPV) infection by granuloviruses (GVs) is well documented; and a GV granule protein, named viral enhancin, has been identified as an active contributor to this effect. We detected the presence of two proteins with molecular mass of 93 and 108 kDa in granules of a GV isolated from Xestia c-nigrum (L.) (XecnGV) as candidates for enhancin, and we confirmed that at least the 108-kDa protein enhances the infectivity of Mamestra brassicae nucleopolyhedrovirus (MabrNPV). We tested the effect of virion-free proteins obtained from XecnGV granules (GVPs) on MabrNPV infection, and we made a comparison with an enhancing chemical, the stilbene-derived fluorescent brightener Tinopal. Bioassay was performed employing the diet contamination method, by using second instars of Mamestra brassicae (L.) (Lepidoptera: Noctuidae). The enhancing effects of GVPs (0.1 mg/g diet) and Tinopal (1 mg/g diet) were estimated to be 70.7–81.5-fold and 26.9–33.7-fold, respectively, as calculated from the LC50 values of MabrNPV with or without the additives. The additives reduced the lethal time of MabrNPV-infected larvae and they caused death at a younger instar. These results suggest that GVPs can enhance MabrNPV infection as effectively as Tinopal.

Mamestra brassicae Nucleopolyhedrovirus Infection and Enhancing Effect of Proteins Derived From Xestia c-nigrum granulovirus in Larvae of Mamestra brassicae and Helicoverpa armigera (Lepidoptera: Noctuidae) on Cabbage

ABSTRACT The insecticidal effect of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) T5 against Mamestra brassicae (L.) and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), important pests of various vegetables and ornamental crops in Japan as well as many other countries, and the enhancing activity of proteins derived from occlusion bodies of Xestia c-nigrum granulovirus (XecnGV) &agr;-4, which was named GVPs, on the infectivity of MabrNPV were evaluated in a bioassay with second-instar larvae fed on virus-applied cabbage, Brassica oleracea L. var. capitata, plants. The lethal concentrations of MabrNPV achieving 95% mortality (LC95) were estimated to be 7.7 × 105 and 1.8 × 105 OBs per ml for M. brassicae and H. armigera, respectively, with MabrNPV-alone treatment. Because the mean areas of cabbage leaf consumed by one larva in 60 h were not significantly different between M. brassicae and H. armigera, we conclude that MabrNPV is more infectious to H. armigera than to M. brassicae. When larvae were fed on cabbage plants treated with 104 OBs per ml MabrNPV and various concentrations of the GVPs, the mortality of the two noctuid larvae increased in relation to GVP concentration. The requisite concentrations of the GVPs achieving 95% mortality with the MabrNPV for M. brassicae and H. armigera were estimated to be 5.93–8.30 and 1.94–3,48 µg/ml, respectively, In a comparison of the MabrNPV-alone treatment with equivalent 95% mortality, addition of GVPs increased the rate of larval death at younger instars, especially in M. brassicae. Our results indicate that GVPs are a potentially useful additive for improving the insecticidal efficacy of MabrNPV.

Estimated time frame for successful PCR analysis of diamondback moths, Plutella xylostella (Lepidoptera: Plutellidae), collected from sticky traps in field conditions

PCR is a useful technique to detect mutations in insecticide resistance genes or to analyze gene flow in pest insects. Plutella xylostella (L.) is one of the most important pests that develops insecticide resistance, and it is desirable to make efficient use of specimens obtained from pheromone-baited sticky traps. To estimate the period of DNA preservation in moths collected on traps in the field, we analyzed the probability of PCR success using mtCOI and microsatellite primers. The results suggested that moths caught by sticky traps would maintain DNA of sufficient quality for PCR analysis when the trap was changed weekly. DNA degradation was greater in moth specimens on traps exposed to ultraviolet (UV) radiation in direct sunlight than in the shade. DNA degeneration was greater in moths exposed to direct sunlight in summer than in winter. Therefore, setting traps in shade or using a UV light-shielding barrier over the trap might be an effective approach to prevent DNA degradation.

Two Year Field Study to Evaluate the Efficacy of Mamestra brassicae Nucleopolyhedrovirus Combined with Proteins Derived from Xestia c-nigrum Granulovirus

Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs) of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV), and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL) to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%). In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL) did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides.