Feeding Behavior Comparison of Bean Bugs, Riptortus pedestris and Halyomorpha halys on Different Soybean Cultivars.

Riptortus pedestris (Fabricius) and Halyomorpha halys (Stål) are the major pests that feed on soybean pods, seeds, and fruits. Higher populations and damage occur during the soybean maturity stages (podding to harvest). To compare the feeding behavior of R. pedestris and H. halys, we used the six most cultivated cultivars (Daepung-2ho, Daechan, Pungsannamul, Daewon, Seonpung, and Seoritae) in Korea using the electropenetrography (EPG) technique. Both R. pedestris and H. halys, the NP (non-penetration), a non-probing waveform, was the shortest in the Pungsannamul (298 and 268 min) and the longest in the Daepung-2ho (334 and 339 min), respectively. The feeding waveforms Pb (phloem feeding: E1-Salivation and E2-Sap feeding) and G (xylem feeding) were the longest in Pungsannamul and the shortest in Daepung-2ho. In addition, as a result of investigating the damage rate by planting six cultivars of beans in the field, as expected, the proportions of damage types B and C were highest in Pungsannamul and lowest in Daepung-2ho. These results reveal that both bug species ingest xylem sap from leaflets and stems using a salivary sheath strategy to acquire water and nutrients from soybean pods/seeds with cell-rupture tactics. This study provides perceptive information to understand the feeding behavior, field occurrence, and damage patterns of R. pedestris and H. halys, which may have key implications for the management of hemipteran pests by determining the specificity and susceptibility of host plants.

Analysis of the Feeding Behavior and Life Table of Nilaparvata lugens and Sogatella furcifera (Hemiptera: Delphacidae) under Sublethal Concentrations of Imidacloprid and Sulfoxaflor.

The brown planthopper (BPH) Nilaparvata lugens and white-backed planthopper (WBPH) Sogatella furcifera are serious rice insect pests that cannot overwinter in Korea and migrate from southeast Asian countries and China. In this study, we investigated the sublethal effects of imidacloprid and sulfoxaflor on the biological parameters and feeding behavior of planthoppers. These sublethal concentrations significantly decreased the net reproduction rate (R0), the intrinsic rate of increase (rm), and the mean generation time (T). For BPHs, the total durations of nonpenetration (NP) waveform by imidacloprid (LC10 = 164.74 and LC30 = 176.48 min) and sulfoxaflor (LC10 = 235.57 and LC30 = 226.93 min) were significantly different from those in the control group (52.73 min). In addition, on WBPHs, the total durations of NP waveform by imidacloprid (LC10 = 203.69 and LC30 = 169.9 min) and sulfoxaflor (LC10 = 134.02 and LC30 = 252.14 min) were significantly different from those in the control group (45.18 min). Moreover, the LC10 and LC30 of these insecticides significantly decreased the phloem feeding time. In conclusion, imidacloprid had a better effect on the inhibition of feeding of the WBPH, and sulfoxaflor showed a better effect on the inhibition of feeding of the BPH. Therefore, it is suggested that treatment with sublethal concentrations of the above insecticides will reduce the feeding of BPHs and WBPHs on rice phloem.

Synergistic Effect of Cold Treatment Combined with Ethyl Formate Fumigation against Drosophila suzukii (Diptera: Drosophilidae).

Drosophila suzukii is a quarantine pest that is rapidly spreading in berries. This study evaluated the synergistic effect of combination treatment with ethyl formate (EF) and cold temperature for D. suzukii control on imported grapes. A higher insecticidal effect was observed at 1 °C than at 5 °C at all developmental stages, and the pupal stage showed the strongest tolerance to cold temperature. After EF fumigation alone, eggs showed the highest tolerance at 216.67 mg·h/L (LCT99 value), and adults showed the highest susceptibility at <27.24 mg·h/L. Among the combination treatment methods, cold temperature after fumigation resulted in the best synergistic effect. The effect of this combination was significant, with 23.3% higher mortality for eggs, 22.4% for larvae, and 23.4% for pupae than observed with EF fumigation alone. Furthermore, the period of complete D. suzukii control in the 12 L desiccator was shorter in the combination treatment group at the LCT80 value than at the LCT50 value of the egg stage. EF showed a very high sorption rate (24%) after 4 h of exposure at a grape loading ratio of 15% in a 0.65 m3 fumigation chamber. As the grape loading ratio for combination treatment decreased, D. suzukii mortality increased, but when EF was administered at the LCT80 value, there was little difference in the mortalities of the eggs and larvae but not the pupae. All D. suzukii developmental stages were completely controlled within 7 days after combination treatment, and phytotoxicity was not observed in grapes. These results suggest that the combination of cold-temperature treatment and EF fumigation could be used for D. suzukii control.

Susceptibility to Acaricides and the Frequencies of Point Mutations in Etoxazole- and Pyridaben-Resistant Strains and Field Populations of the Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

The two-spotted spider mite Tetranychus urticae Koch is a major agricultural pest worldwide and is known to rapidly develop resistance to pesticides. In the present study, we explored a field strain that was collected in 2000 and 2003 and has been exhibiting resistance to etoxazole and pyridaben over the last 16 years. The resistance ratios of the etoxazole- and pyridaben-resistant strains (ER and PR) to etoxazole or pyridaben were more than 5,000,000- and 4109.6-fold higher than that of the susceptible strain, respectively. All field-collected populations showed resistance to etoxazole and pyridaben. The ER and PR strains showed cross-resistance to several acaricides. Both I1017F and H92R point mutations were detected in 7 out of 8 field groups. Spirodiclofen and spiromesifen resulted in more than 77.5% mortality in the 8 field groups. In addition, the genotype frequency of the I1017F point mutation was 100.0% in the ER strain, and that of the H92R point mutation was 97.0% in the PR strain. All of the field populations were found to have a high frequency of I1017F. These results suggest that the observation of resistance patterns will help in designing a sustainable IPM program for T. urticae.

Fumigation Activity against Phosphine-Resistant Tribolium castaneum (Coleoptera: Tenebrionidae) Using Carbonyl Sulfide.

Phosphine resistance is occurring among stored-grain pests worldwide. This study investigated the fumigation activity of phosphine (PH3) and carbonyl sulfide (COS) against domestic strain (d-strain) Tribolium castaneum, resistance strain (r-strain) T. castaneum and Oryzaephilus surinamensis. All developmental stages of the pests were exposed to two fumigants (PH3 and COS), and the fumigation activity according to the dose and exposure time was evaluated in a 12-L desiccator and 0.5 m3 fumigation chamber. The rice sorption rate and quality following exposure to thetwofumigants were evaluated. The mortality was 2.9% in r-strain T. castaneum, 49.5% in d-strain T. castaneum and 99.2% in O. surinamensis when 2 mg/L PH3 was used in a 12-L desiccator for 4 h. However, all pest developmental stages showed 100% mortality after 24 h of exposure in the 0.5 m3 fumigation chamber, except for the r-strain T. castaneum. A mortalityof 100% was observed in all of the r-strain T. castaneum developmental stages at an exposure time of 192 h. For COS applied at 40.23 mg/L and 50 g/m3 in the 12-L desiccator and the 0.5 m3 fumigation chamber, respectively, 100% mortality was observed across all developmental stages regardless of species and strain. The sorption of COS was 10% higher than that of PH3, but there was no significant difference in rice quality compared to that in the control. Therefore, this study suggests that COS can be used for controlling T. castaneum resistant to PH3.

Target-Site Mutations and Glutathione S-Transferases Are Associated with Acequinocyl and Pyridaben Resistance in the Two-Spotted Spider Mite Tetranychus urticae (Acari: Tetranychidae).

The two-spotted spider mite Tetranychus urticae is a difficult-to-control pest due to its short life cycle and rapid resistance development. In this study, we characterized field strains collected in 2001 and 2003 that were selected for acequinocyl resistance (AR) and pyridaben resistance (PR), respectively. These strains displayed resistance ratios of 1798.6 (susceptible vs. AR) and 5555.6 (susceptible vs. PR), respectively, and were screened for cross-resistance against several currently used acaricides. The AR strain exhibited pyridaben cross-resistance, but the PR strain showed no cross-resistance. The AR strain exhibited point mutations in cytb (I256V, N321S) and PSST (H92R). In contrast, the PR strain exhibited the H92R but not the I256V and N321S point mutations. In some cases increased glutathione S-transferase (GST) activity has previously been linked to enhanced detoxification. The AR strain exhibited approximately 2.3-, 1.8-, and 2.2-fold increased GST activity against 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and 4-nitrobenzyl chloride (NBC), respectively. Among the five GST subclass genes (delta, omega, mu, zeta, and kappa), the relative expression of delta class GSTs in the AR strain were significantly higher than the PR and susceptible strain. These results suggest that the I256V and N321S mutations and the increased GST metabolism and GST delta overexpression might be related to acequinocyl resistance in T. urticae.

Control of Whitefly (Hemiptera: Aleyrodidae), Trialeurodes vaporariorum, with Electron Beam and X-Ray Radiation of Fresh Strawberies for Export.

Strawberry (Fragaria ananassa Duch) is one of the representative fresh agricultural products exported overseas from South Korea. The greenhouse whitefly (Hemiptera: Aleyrodidae), Trialeurodes vaporariorum, is an economically important insect pest of commercial strawberries in South Korea. The objective of the present study was to evaluate the effects of electron beam and X-ray on the development and reproduction of T. vaporariorum. To determine the radiation dose as a quarantine treatment for strawberry, T. vaporariorum were placed at the top, middle, and bottom location in boxes filled with strawberry fruits and irradiated. Eggs were completely inhibited from hatching at 50 Gy, and adult emergence of 3rd nymphs was completely suppressed at 150 Gy in both electron beam and X-ray. Some adults spawning occurred at 100 Gy. However, F1 hatchability was completely suppressed. The results suggest that T. vaporariorum was the most radiotolerant to both of ionization energy at the nymph stage. The dosimetry results showed that the penetrating power of ionizing radiation in boxes filled with strawberry fruits was the lowest at the bottom location. A treatment dose of 150 Gy is adaptable as a quarantine treatment to T. vaporariorum nymph in strawberry fruit. Our results indicate that ionizing radiation could be recommendable as a phytosanitary treatment for quarantine.

Gamma-Ray Irradiation Control of Whiteflies Bemisia tabaci (Hemiptera: Aleyrodidae) and Trialeurodes vaporariorum in the Exportation of Fresh Strawberries.

Whitefly pests, including the sweetpotato whitefly, Bemisia tabaci (Gennadius), and the greenhouse whitefly, Trialeurodes vaporariorum (Westwood), are economically important in agriculture. With the annual growth of the domestic fresh fruit export market, various quarantine treatment methods are being used to export strawberries of better quality. The objective of the present study was to evaluate the effects of gamma rays on the development and reproductive sterility of B. tabaci and T. vaporariorum. In both species, the eggs were completely inhibited from hatching at 50 Gy, and the emergence of third-instar nymphs was completely suppressed at 150 Gy. Some adult B. tabaci and T. vaporariorum spawning occurred at 100 and 70 Gy, respectively; however, at these irradiation levels, F1 hatchability was completely inhibited. Dosimetry results showed that the penetrating power of gamma ray in the strawberry-filled box was the lowest at the mid-box position. Therefore, B. tabaci and T. vaporariorum were placed in the middle of the strawberry-filled box and irradiated. A gamma-ray irradiation of 100 Gy suppressed the development and reproduction of eggs and adults in both B. tabaci and T. vaporariorum. Our data suggest that at least 100 Gy should be used for the control of these two species of whitefly for strawberry export.

Comparison of the Efficacy and Phytotoxicity of Phosphine and Ethyl Formate for Controlling Pseudococcus longispinus (Hemiptera: Pseudococcidae) and Pseudococcus orchidicola on Imported Foliage Nursery Plants.

The fumigation activity of phosphine (PH3) and ethyl formate (EF) and their phytotoxicity to 13 imported foliage nursery plant species were evaluated. The lethal concentration and time (LCT99) values of the PH3 indicated that the susceptibility of the nymphs (3.95 and <0.45 mg·h/liter, respectively) was higher than that of the adults (5.29 and 3.66 mg·h/liter, respectively) of two mealybugs [Pseudococcus longispinus (Targioni-Tozzetti) and P. orchidicola Takahashi]. The highest concentration reduction rate of PH3 and EF on the 13 foliage nursery plants in the 12-liter desiccator was 41.5% for Heteropanax fragrans and 71.7% for Schefflera arboricola, respectively, which indicates that PH3 has a lower sorption rate than EF. The phytotoxicities of PH3-treated foliage nursery plants did not significantly differ from those of the nontreated plants, but EF caused phytotoxicity in 11 foliage nursery plants a week after treatment. When the exposure time of PH3 increased to 24 h, the adults and nymphs of both mealybug species showed 100% mortality in the 0.5 m3 fumigation chamber. In the 10 m3 fumigation container used in the field, there was 100% mortality of both mealybugs after treatment with 2 g/m3 PH3 for 24 h at 16°C. These results indicate that EF is not a suitable mealybug fumigant due to its high sorption and phytotoxicity to foliage nursery plants, despite fumigation activity against the two species. However, PH3 seems to be suitable for mealybug fumigation in foliage nursery plants and can be used as a substitute for methyl bromide.

Acequinocyl Resistance Associated With I256V and N321S Mutations in the Two-Spotted Spider Mite (Acari: Tetranychidae).

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most important pest species, because it devastates many horticultural and ornamental crops and fruit trees. In the present study, we explored a field strain that was collected in January 2001 and then selected for 16 years for acequinocyl resistance. The resistance ratios calculated for the LC50 value in the laboratory-selected acequinocyl-resistant (LSAR16) strain was 4,237-fold higher than that of the susceptible strain. Pretreatment with the synergists piperonyl butoxide and S,S,S-tributyl-phosphorotrithioate significantly increased the toxicity of acequinocyl to the LSAR16 strain. Crossing experiments revealed that the resistance in the LSAR16 strain was maternally inherited, dominant, and monogenic. Furthermore, among individuals in the LSAR16 strain, 85.5-98.5% had the I256V mutation and 98-99% had the N321S mutation in mitochondrial cytochrome b. These results suggest that these two new point mutations contribute to acequinocyl resistance in T. urticae.

Elucidation of molecular expression associated with abnormal development and sterility caused by electron beam irradiation in Spodoptera litura (F.) (Lepidoptera: Noctuidae).

PURPOSE: The objective of the present study was to elucidate the mode of indirect action of electron beam irradiation at the molecular level against a quarantine pest, Spodoptera litura (F.). MATERIAL AND METHODS: Electron beam irradiation (50-200 Gy) was applied to S. litura eggs, larvae, pupae, and adults, after which the feeding area, body weight, deformity of pupae and adults, ovarian development, expression levels of vitellogenin (Vg) and vitellogenin receptor (VgR) genes, and protein levels were analyzed. RESULTS: The amount of feeding by S. litura larvae and the synthesis level of 70 kDa storage protein significantly decreased as the electron beam dose increased. When larvae were treated with the electron beam, morphological deformities appeared in the pupae, and abnormal wing disc (AWD) expression significantly decreased. Ovarian development was completely inhibited in emerged adults that had undergone 200 Gy electron beam irradiation as pupae. Quantitative real-time PCR (qRT-PCR) assays showed significant downregulation of the Vg and VgR genes due to electron beam irradiation; whereas the synthesis level of Vg protein (190 kDa) did not decrease with time in eggs unlike in non-irradiated (control) S. litura eggs, exhibiting irradiation induced impairment of Vg functioning. CONCLUSIONS: These findings of radiation-induced abnormal development and sterility in S. litura together with the correlated changes at the molecular level may facilitate the development of a phytosanitary strategy against this quarantine pest using electron beam irradiation.

Efficacy and Phytotoxicity of Phosphine as Fumigants for Frankliniella occidentalis (Thysanoptera: Thripidae) on Asparagus.

The insecticidal activity of phosphine (PH3) and ethyl formate (EF) toward Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) and their phytotoxicity to asparagus were evaluated. Both the PH3 and EF fumigants showed higher lethal concentration and time (LCT) values at lower temperatures. The LCT99 values of PH3 and EF at 5°C in a 12 liters desiccator for 4 h showed the following ranking: eggs (64.69 mg·h/liter for PH3 and EF indicating phytotoxicity to asparagus), nymphs (5.54 and 17.48 mg·h/liter, respectively), and adults (3.83 and 14.67 mg·h/liter, respectively). The adsorption of PH3 was approximately 11% at 2°C and 13% at 5°C, whereas the adsorption of EF increased sharply to 88% at 2°C and 97% at 5°C. The hatching rate of F. occidentalis eggs was approximately 95% at all locations (top, middle, and bottom) in the presence of 4 mg/liter PH3 at 5°C in a 0.65-m3 fumigation chamber for 24 h. However, extension of the treatment to 48 h resulted in 100% inhibition of egg hatching. The atmospheric level of PH3 decreased below the threshold limit value after 80 min, and phytotoxicity was not observed. The results revealed that EF is highly absorbed by asparagus and is not suitable as a fumigant, but PH3 is a suitable alternative to the fumigant methyl bromide for the control of western flower thrips in asparagus.

Fumigation Activity of Ethyl Formate and Phosphine Against Tetranychus urticae (Acari: Tetranychidae) on Imported Sweet Pumpkin.

This study investigated an alternative fumigant for imported sweet pumpkin. Laboratory fumigations with ethyl formate (EF) and phosphine (PH3) alone and in combination were conducted at different temperatures in 12-liter desiccators to determine their efficacy and synergism against adults and eggs of two-spotted spider mites (Tetranychus urticae Koch), a major pest of sweet pumpkins; larger scale fumigations with EF were also performed to evaluate the impact of EF fumigation on the postharvest quality of sweet pumpkin and EF absorption. EF fumigation of 4 h achieved 100% control of both adults and eggs at 10°C. Efficacy of EF also increased with temperature. Conversely, PH3 fumigation for 4 and 6 h was not effective against either adults or eggs, and no synergism between EF and PH3 was observed. In larger scale fumigation tests, EF at 60 g/m3 for 4 h resulted in 100% control of both adults and eggs at 10°C, without causing injury to the sweet pumpkins. These results suggest that EF has the potential to be a safe and effective alternative to methyl bromide fumigation for controlling two-spotted spider mites on sweet pumpkin.

Electron Beam-Induced Sterility and Inhibition of Ovarian Development in the Sakhalin Pine Longicorn, Monochamus saltuarius (Coleoptera: Cerambycidae).

The Sakhalin pine longicorn, Monochamus saltuarius (Gebler; Coleoptera: Cerambycidae), is an insect vector of the pine wilt nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, and is widely distributed in central Korea. M. saltuarius is a forest pest that seriously damages Pinus densiflora (Siebold et Zucc, Pinales: Pinaceae) and Pinus koraiensis (Siebold & Zucc, Pinales: Pinaceae) forests. We examined the effect of electron beam irradiation on the mating, DNA damage and ovarian development of M. saltuarius adults and sought to identify the optimal dose for sterilizing insects. When the adults were irradiated with electron beams, both females and males were completely sterile at 200 Gy. In a reciprocal crossing experiment between unirradiated and irradiated adults, the reproductive ability of wild adults was recovered by crossing with wild adults even after crossing previously with sterile adults. When a pair of unirradiated adults (♀- × â™‚-) and 10 or 20 irradiated adults (♀+ or ♂+) were kept together, the control effect was as high as 80~90%. After electron beam irradiation at 200 Gy, the DNA of M. saltuarius adults was damaged, the ovarian development of female adults was inhibited, and the level of vitellogenin was significantly decreased compared with that in unirradiated female adults. These results suggest that pine wilt disease can be effectively controlled if a large number of sterilized M. saltuarius male adults are released into the field.

X-ray Irradiation Control of Frankliniella occidentalis and Frankliniella intonsa (Thysanoptera: Thripidae) in the Exportation of Freshly Cut Lily Flowers.

Lily (Lilium longiflorum Thunb.) is the most representative bulb flower, and it is the third most important flower in the flower industry of South Korea after rose and chrysanthemum. To determine the efficacy of X-ray irradiation for use in quarantine processing, two species of flower thrips (Frankliniella intonsa (Trybom) and Frankliniella occidentalis (Pergande)) were placed in the top, middle, and bottom locations of lily boxes and irradiated with different X-ray doses. After irradiation with an X-ray dose of 150 Gy, the egg hatching of the two flower thrips was completely inhibited at every location in the lily boxes, and the irradiated F. intonsa and F. occidentalis nymphs failed to emerge as adult in every location of the lily boxes. When the adults were irradiated at 150 Gy, the fecundity of the two flower thrips was markedly lower than that of the untreated control groups. The F1 generation failed to hatch at the top and middle locations, whereas the F1 generation of both F. intonsa and F. occidentalis was not suppressed at the bottom locations, even at 200 Gy. However, hatching was perfectly inhibited at 300 Gy of X-ray irradiation. Also, X-rays did not affect the postharvest physiology of cut lilies. Therefore, a minimum dose of 300 Gy is recommended for the control of F. intonsa and F. occidentalis for the exportation of lily.

Comparative Analyses of Cu-Zn Superoxide Dismutase (SOD1) and Thioredoxin Reductase (TrxR) at the mRNA Level between Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) Under Stress Conditions.

This study compared stress-induced expression of Cu-Zn superoxide dismutase (SOD1) and thioredoxin reductase (TrxR) genes in the European honeybee Apis mellifera L. and Asian honeybee Apis cerana F. Expression of both SOD1 and TrxR rapidly increased up to 5 h after exposure to cold (4 °C) or heat (37 °C) treatment and then gradually decreased, with a stronger effect induced by cold stress in A. mellifera compared with A. cerana. Injection of stress-inducing substances (methyl viologen, [MV] and H2O2) also increased SOD1 and TrxR expression in both A. mellifera and A. cerana, and this effect was more pronounced with MV than H2O2. Additionally, we heterologously expressed the A. mellifera and A. cerana SOD1 and TrxR proteins in an Escherichia coli expression system, and detection by SDS-PAGE, confirmed by Western blotting using anti-His tag antibodies, revealed bands at 16 and 60 kDa, respectively. Our results show that the expression patterns of SOD1 and TrxR differ between A. mellifera and A. cerana under conditions of low or high temperature as well as oxidative stress.

Synergistic Effects of Oxygen on Phosphine and Ethyl Formate for the Control of Phthorimaea operculella (Lepidoptera: Gelechiidae).

Phosphine (PH3) and ethyl formate (EF) are two potentially powerful postharvest fumigant insecticides. We investigated the effectiveness of both PH3 and EF as fumigants at all developmental stages of the potato tuber moth Phthorimaea operculella Zeller, and we also studied the synergistic effects of these fumigants under controlled atmospheres of 50 and 80% oxygen (O2). The larval stage of P. operculella was the most susceptible to fumigation with PH3 at both 5°C and 20°C. All of the developmental stages showed greater susceptibility to PH3 at 20°C than at 5°C, whereas the susceptibility of adult P. operculella to this fumigant was not affected by temperature. The toxicity of EF did not differ with temperature for any of the P. operculella developmental stages. The atmospheric oxidation of PH3 increased the toxicity of this fumigant toward all developmental stages at both temperatures. In contrast, no differences in toxicity were observed for oxidized EF compared with EF alone at any developmental stage. In conclusion, using fumigation tests, we showed that atmospherically oxidized PH3 was much more effective against P. operculella than PH3 alone, demonstrating a synergistic effect for this fumigant and O2. Therefore, treatment with PH3 and high concentrations of O2, as described in this study, could be useful for managing the postharvest pest P. operculella.

Hyper-enhanced production of foreign recombinant protein by fusion with the partial polyhedrin of nucleopolyhedrovirus.

To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus.

Production of classical swine fever virus envelope glycoprotein E2 as recombinant polyhedra in baculovirus-infected silkworm larvae.

Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg/ml of hemolymph and 0.53 mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen.

Chemical composition, larvicidal action, and adult repellency of Thymus magnus against Aedes albopictus.

Thymus magnus, an endemic species, is found in the Republic of Korea. The volatile compounds extracted by SPME from T. magnus were investigated for their chemical composition and electrophysiological response against the Asian tiger mosquito, Aedes albopictus. The volatile compounds of T. magnus as determined by gas chromatography mass spectrometry were gamma-terpinene (33.0%), thymol (29.9%), beta-bisabolene (8.9%), p-cymene (8.3%), alpha-terpinene (5.0%), myrcene (4.7%), beta-caryophyllene (4.0%), alpha-thujene (2.7%), camphene (1.3%), carvacrol (1.2%), and alpha-pinene (1.1%). Among these candidates, thymol exhibited complete (100%) repellent activity against female Ae. albopictus, an effect that was confirmed through evaluating the electrophysiological response on the antenna of Ae. albopictus. The effectiveness of a binary 1:2 mixture of thymol and vanillin (0.05:0.1 microl per cm2) was found to be significantly more effective than thymol alone for a period of 120 min. In addition, thymol, alpha-terpinene, and carvacrol showed high larvicidal activity against on the third-stage larvae with LC50 values of 0.9 microl per 100 ml.