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.     

Foraging Ants as Scavengers on Entomopathogenic Nematode-Killed Insects

Ants were the most apparent invertebrate scavengers observed foraging on entomopathogenic nematode-killed insects (i.e., insect cadavers containing entomopathogenic nematodes and their symbiotic bacteria) in the present study. Workers of the Argentine ant,Linepithema humile(Mayr), scavenged nematode-killed insects on the surface and those buried 2 cm below the soil surface. Ant workers scavenged significantly more steinernematid-killed (60–85%) than heterorhabditid-killed (10–20%) insects. More 4-day-postinfected cadavers (hosts died within 48 h after exposure to nematodes) were scavenged than 10-day-postinfected cadavers. Ten-day-postinfected hosts contained live infective juvenile nematodes therefore ants may serve as phoretic agents. Other ant species, includingVeromessor andrei(Mayr),Pheidole vistanaForel,Formica pacificaFrancoeur, andMonomoriom ergatogynaWheeler, also scavenged nematode-killed insects. These ant species removed or destroyed about 45% of the steinernematid-killed insects. These results suggest that survival of steinernematid nematodes may be more significantly impacted by invertebrate scavengers, especially ants, than that of heterorhabditid nematodes, and placement of steinernematid-killed insects in the field for biological control may be an ineffective release strategy. Because entomopathogenic nematodes kill insects with the help of symbiotic bacteria, we tested the role of these bacterial species in deterring invertebrate scavengers by injecting bacteria (without nematodes) into insects and placing the cadavers in the field. None of the insects killed by the symbiotic bacterium,Photorhabdus luminescens(Thomas and Poinar) fromHeterorhabditis bacteriophoraPoinar, were scavanged, whereas 70% of the insects killed by the symbiotic bacterium,Xenorhabdus nematophilus(Poinar and Thomas) fromSteinernema carpocapsae(Weiser), and 90% of the insects killed byBacillus thuringiensisBerliner were scavenged by the Argentine ant. We conclude thatP. luminescensis responsible for preventing ants from foraging on heterorhabditid-killed hosts.     

Phenotypic characteristics and relative proportions of three genotypic variants isolated from a nucleopolyhedrovirus of Spodoptera exigua

US2A, US2D, and US2F are three in vivocloned genotypic variants from the wild-type strain of a Spodoptera exiguanucleopolyhedrovirus (SeMNPV) isolated in Florida (USA) and is the active component of the commercial bioinsecticide Spod-X^®. These variants were compared in terms of pathogenicity (LD₅₀), speed of kill (expressed as mean time to death) and viral progeny productivity (OBs/larva). LD₅₀values were similar for the three cloned genotypes. The mean time to death value for US2D (113.7 h) was significantly higher than those of US2A (31.7 h) and US2F (27.8 h). Virus yield was determined for L₄larvae infected with the estimated LD₉₉. US2D infected larvae attained higher weight than those infected with US2A and US2F, and produced a higher OB yield than larvae infected with US2A or US2F. An outstanding feature of US2F, in contrast to US2A and US2D, was its inability to disrupt the teguments of NPV-killed larvae. To study the relative proportion of the three genotypic variants throughout successive passages, S. exigualarvae were originally infected with a viral inoculum containing a 1:1:1 mixture of the three genotypes. After three successive passages, US2D was no longer detected in either of the three replicate experiments performed, while US2A was the predominant genotype in all of them, and US2F remained at similar proportions throughout the three passages. The influence of the phenotypic characteristics of the three variants on their relative proportions in mixed infections is discussed.     

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.     

Juvenile hormone analogs greatly increase the production of a nucleopolyhedrovirus

The commercial production of baculovirus insecticides is limited by the need to produce the virus in living insects. The influence of juvenile hormone analogs (JHA) on the growth and survival of Spodoptera exigua larvae placed on treated diet in the fifth instar was examined. Weight increases observed in methoprene- and fenoxycarb-treated larvae were over three-fold greater than that of control insects, whereas other compounds resulted in lower weight gains (pyriproxyfen) or highly variable responses (hydroprene). Approximately 90% and 70% of fenoxycarb and methoprene-treated larvae, respectively, molted to a supernumerary sixth instar and attained a final weight at 8–10 days post-treatment that was approximately double the maximum weight observed in control larvae. Inoculation of fenoxycarb and methoprene-treated sixth instars with a nucleopolyhedrovirus (SeMNPV) resulted in 2.4- or 2.9-fold increases in final weights, compared to control larvae inoculated in the fifth instar. The total yield of SeMNPV occlusion bodies (OBs) per larva was 2.7- and 2.9-fold greater in fenoxycarb- and methoprene-treated larvae, respectively, compared to fifth instar controls. A significant but small increase in the yield of OBs/mg larval weight was observed in fenoxycarb-treated insects but not in the methoprene treatment. The LC₅₀ value of OBs harvested from fenoxycarb-treated insects was slightly higher than that of OBs from control insects, whereas no such difference was observed in OBs from methoprene-treated insects. We conclude that appropriate use of JHA technology is likely to provide considerable benefits for the mass production of baculoviruses.     

Chemical and biological stress factors on the activation of nucleopolyhedrovirus infections in covertly infected Spodoptera exigua

Following the consumption of baculovirus occlusion bodies (OBs), insects may succumb to lethal disease, but the survivors can harbour sublethal covert infections and may develop, reproduce and transmit the infection to their offspring. The use of different chemical and biological stressors was examined to determine whether they could be used to activate covert infections in populations of Spodoptera exigua larvae infected by the homologous nucleopolyhedrovirus (SeMNPV). Treatment of covertly infected S. exigua second instars with Tinopal UNPA‐GX, hydroxylamine, paraquat, Bacillus thuringiensis var. kurstaki crystals, spores or mixtures of crystals + spores, or a heterologous nucleopolyhedrovirus (Chrysodeixis chalcites SNPV) did not result in the activation of SeMNPV covert infections. Similarly, virus treatments involving permissive NPVs did not result in greater mortality in covertly infected insects compared with the virus‐free controls. In contrast, 0.1% copper sulphate, 1% iron (II) sulphate and 1 mg/l sodium selenite treatments resulted in 12–41% lethal polyhedrosis disease in covertly infected larvae. A greenhouse trial using copper sulphate and sodium selenite as activation factors applied to covertly infected S. exigua larvae on sweet pepper plants resulted in very low levels of SeMNPV activation (<3%). These results highlight the important roles of copper, iron and selenium in insect immunity and baculovirus‐induced disease. However, these substances seem unlikely to prove useful for the activation of covert SeMNPV infections in S. exigua larvae under greenhouse conditions.     

ANALYSIS OF HELICOVERPA ARMIGERA SINGLE NUCLEOPOLYHEDROVIRUS IMMEDIATE EARLY 1 (IE‐1) GENE*

Abstract A 6.12 kb Xbal‐H fragment of the Helicoverpa armigem single nucleopolyhedrovirus (HaSNPV) gemone was cloned and the complete sequence of this fragment was sequenced by random sequencing method. Sequence comparison and analysis revealed an ORF13 which was homologous to ie‐1 of Auiographa California nucleopolyhedrovirus (AcMNPV). The homologous encoding gene is ie‐1. The total length of the encoding region of HaSNPV gene was 1986 bp and was predicted to encode 661 amino acid protein(IE‐1) with molecular weight of 76.5 kD. The alingment of putative HaSNPV IE‐1 amino acid sequence with those of other 9 reported baculoviruses IE‐Is showed that the HaSNPV IE‐1 was most closely related to Helicoverpa zea nucleopolyhedrovirus (HzNPV) IE‐1, with 97% amino acid identidy. But it showed a low degree of sequence similarity to those of AcMNPV, Bombyx mori nucleopolyhedrovirus (BmNPV), Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV), Lymantria dispar nucleopolyhedrovirus (LdMNPV), Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV), Spodoptera exigua nucleopolyhedrovirus (SeMNPV), Plutella xylostella granulovirus(PxGV) and Xestia c‐nigrum granulovirus (XcGV), with 23%, 23%, 23%, 25%, 23%, 14%, 27% and 7% amino acid identity, respectively. A phylogenetic tree of ten baculoviruses IE‐1 was also given.     

Signal Sequence and Promoter Effects on the Efficacy of Toxin-Expressing Baculoviruses as Biopesticides

Baculovirus recombinants expressing a neurotoxin gene,tox34,from the straw itch mitePyemotes triticihave been previously shown to paralyze or kill insects approximately 50% faster than wild-type. We constructed a series of recombinants of the baculovirusAutographa californicanucleopolyhedrovirus which expressedtox34with different signal sequences or were controlled by different promoters to evaluate their influence on toxin expression in cell culture and in insects. Heterologous signal sequences provided no significant increase in the overall levels of the maturetox34gene product, Tox34, secreted into the tissue culture media from infected cells and no improvement in the time required for paralysis of insect hosts. The time required for paralysis was promoter-dependent; the late 6.9K DNA binding protein gene promoter was generally the most effective promoter, although an insect HSP70 promoter was equally or more effective in one of the species.     

Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua

Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82–93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen.     

In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera)

Summary The in vitro host range of a newly isolated baculovirus from the diamondback moth Plutella xylostella was tested against six lepidopteran cell lines. Two baculoviruses with host ranges from the alfalfa looper Autographa californica (A. californica multiple nucleopolyhedrovirus, AcMNPV) and the celery looper Anagrapha falcifera (AfMNPV) were also included in this study for comparative purposes. PxMNPV replicated in all six cell lines and produced occlusion bodies, with HV-AMI and TN-CLI cells producing the highest viral titers and greatest number of occlusion bodies. There was no significant replication of AcMNPV and AfMNPV in the HZ-FB33 cell line and thus no production of occlusion bodies. The restriction endonuclease profiles of the three baculoviruses showed similarities but could be readily distinguished from each other. Either HV-AM1 or TN-CL1 would be suitable cell lines for the in vitro production of PxMNPV.     

The survival ofZoophthora radicans (Zygomycetes: entomophthorales) isolates as hyphal bodies in mummified larvae ofPlutella xylostella (Lep.: Yponomeutidae)

The survival of three isolates ofZoophthora radicans (NW 250, NW 253 & NW 182) as hyphal bodies in dried larvae ofPlutella xylostella stored at 4, 10 and 20°C and 20% R.H was determined. After storage at 20°C, the production of conidia by all isolates was unaffected after 2 weeks but diminished increasingly after 4 and 8 weeks and was entirely lost after 16 weeks. By comparison conidium production at 10°C was unaffected after 16 weeks (isolates NW 250 and NW 182) and, 24 weeks (NW 253) of storage though it declined rapidly in all isolates thereafter. At 4°C many conidia were produced by all isolates even after 34 weeks of storage. These results are consistent with work on other entomophthoralean fungi in dried cadavers suggesting that this may be a common survival strategy in these fungi. NW 250, 253 and 182 were isolated fromP. xylostella in Malaysia and Taiwan, where conditions allow the host to remain active throughout the year. None produced resting sporesin vivo orin vitro but as hosts are always available the ability to survive short dry periods is probably more important than long-term survival for which resting spores are most adapted.      

A cell strain cloned from Spodoptera exigua cell line (IOZCAS-Spex-II) highly susceptible to S. exigua nucleopolyhedrovirus infection

A cell strain (IOZCAS-Spex-II-A) cloned from IOZCAS-Spex-II, a cell line established from the fat body of Spodoptera exigua (Lepidoptera: Noctuidae) larva, was characterized, and its capability to produce S. exigua nucleopolyhedrovirus was high with infection rate exceeding 90% compared with its parental cell line IOZCAS-Spex-II that scored only 50%. Growth curve of budded virus (BV) in the strain was analyzed and the titer of BV reached the highest of 3.7?×?10⁴ pfu/mL by 96 h after inoculation. Concentration of occlusion bodies (OBs) produced by the cloned cell strain (IOZCAS-Spex-II-A) was 7.1?×?10⁷ OBs/mL, while the parental cell line produced 2.4?×?10⁷ OBs/mL. The average yield of the virus was 176 OBs/cell of IOZCAS-Spex-II-A compared with 211 OBs/cell that of the parental cell line. Significant differences were observed in virus production, growth characters, cell shape, between the parental cell line, and its clone. The cell lines (IOZCAS-Spex-II and IOZCAS-Spex-II-A) were also susceptible to Autographa californica multiple nucleopolyhedrovirus infection. In addition, they were characterized with regard to their growth rates and DNA amplification fingerprinting technique employing polymerase chain reaction.     

Impact of flowering buckwheat on Lepidopteran cabbage pests and their parasitoids at two spatial scales

We assessed the potential of annual buckwheat, Fagopyrum esculentum Moench, to lead to improved parasitism of lepidopteran cabbage pests over four years. Pest, parasitism, and hyperparasitism rates were monitored in replicated cabbage plots (12 × 20 m) with or without 3 m wide buckwheat borders from 2000 to 2003. Floral borders did not significantly increase egg, larval, or pupal densities of cabbage looper, Trichoplusia ni (Hübner), imported cabbageworm, Pieris rapae (L.), or diamondback moth, Plutella xylostella (L.). Buckwheat increased parasitism rates by Voria ruralis (Fallen) on T. ni larvae and Cotesia rubecula (Marshall) on P. rapaelarvae over four years. Parasitism by Diadegma insulare (Cresson) on P. xylostella larvae was higher in buckwheat than control plots in the first year, and parasitism by Euplectrus plathypenae (Howard) on T. ni larvae was lower in buckwheat than control plots in the second year. The hyperparasitoid Conura side (Walker) attacked D. insulare all four years, but buckwheat did not affect hyperparasitism rates. The effect of spatial scale on pest densities and parasitism in 2001 was evaluated by comparing plots separated at least 67 m (nearby) versus 800 m apart (isolated). T. ni pupae and P. rapae eggs and pupae were more abundant in plots in closer proximity, whereas P. xylostella densities did not vary by the spatial separation of plots. Tachinids and Pteromalus puparum (L.) attacked more P. rapae in nearby plots. E. plathypenae responded to the treatment × scale interaction, parasitizing more in control than buckwheat when plots were isolated but not when plots were nearby.     

Effects of temperature on acaricidal and insecticidal activities of the benzoylureas flucycloxuron and diflubenzuron

Effects of temperature on the activity of flucycloxuron on larval stages of Panonychus ulmi (Koch), based on LC₅₀ values, were highly significant (P < 0.001) with temperature coefficients of-1.7 in both the ranges of 15° to 25°C and 20° to 30°C. The slopes of probit regression lines at 15° and 20°C were significantly steeper than those at 25° and 30°C. As a consequence the temperature coefficients based on LC₉₀ values were-4.4 and-2.2, for the 2 temperature ranges. The ovicidal activity of flucycloxuron on P. ulmi was low and was only statistically detectable at 20°C (LC₉₀ of 84 mg a.i./l). In studies with larvae of Aedes aegypti (Linnaeus), Leptinotarsa decemlineata (Say), Plutella xylostella (Linnaeus), Spodeptera exigua (Hübner) and Spodoptera littoralis (Boisduval) probit regression lines were parallel over temperature. The activity of flucycloxuron on these five insect species was not affected by temperature. Based on LC₅₀ values, diflubenzuron showed positive temperature coefficients on P. xylostella of + 2.1 at 15° to 25°C and + 2.5 at 20° to 30°C. For S. littoralis the temperature coefficient was positive (+ 2.4) at 15° to 25°C but negative (-1.9) at the 20° to 30°C range. Temperature coefficients of diflubenzuron were neutral for A. aegypti, L. decemlineata and S. exigua. In the design and analysis of these studies special allowance was made for date effects and variation in natural mortality over temperature.     

Effects of host age on parasitism and progeny allocation in Trichogrammatidae

The impact of host age on the number of hosts killed, survival of progeny, progeny allocation, and sex allocation was examined for several Trichogrammatidae (Hymenoptera) species in laboratory choice tests. Individual female parasitoids were provided with young, medium-aged and old eggs of one of three lepidopterous host species: Trichoplusia ni (Hübner) (Noctuidae), Pieris rapae (L.) (Pieridae), or Plutella xylostella (L.) (Plutellidae). Trichogrammatid species behaved as gregarious parasitoids with the first two host species, and as solitary parasitoids with eggs of the smaller latter one. They mostly preferred young eggs of T. ni, but did not discriminate among P. rapae eggs of different ages, and often preferred young or medium-aged P. xylostella eggs over old eggs. Survival of progeny did not vary constantly with host age, although it was often very low in P. rapae eggs of any age. Clutch size frequently decreased with host age in both T. ni and P. rapae. Offspring sex ratio did not change with age of T. ni and P. rapae eggs, and rarely did so in P. xylostella eggs. In regard to host age, the results with T. ni are the ones which are the most in agreement with optimal foraging theoretical predictions, as clutch size was the highest in preferred younger eggs.     

Parasitoid Preference for Host-Infested Plants Is Affected by the Risk of Intraguild Predation

Flowering Rorippa indicaplants are attended by ants that collect nectar and, at the same time, prey on herbivorous insects, including larvae of the diamondback moth, Plutella xylostella.Here, we showed that P. xylostellalarvae suffered higher predation on R. indicawhose flowers were accessible by ants than on plants those whose flowers were inaccessible. Ants showed equal predation preference between unparasitized and larvae parasitized by Cotesia plutellae,a dominant specialist parasitic wasp of P. xylostellalarvae. C. plutellaepreferred non-flowering, host-infested R. indicato flowering, host infested R. indica.Based on these results, we infer that the preference of C. plutellaefor non-flowering, host-infested plants is in part explained by the avoidance of intraguild predation by attending ants.     

Occurrence and characterization of a nucleopolyhedrovirus from <Emphasis Type="Italic">Maruca vitrata</Emphasis> (Lepidoptera,Pyralidae) isolated in Taiwan

A nucleopolyhedrovirus (MaviMNPV) was isolated from diseased larvae of legume pod borer (LPB), Maruca vitrata, at Tainan in Taiwan. Electron microscopical studies on the ultrastructure of MaviMNPV occlusion bodies (OBs) showed several virions (up to 19) with multiple nucleocapsids (up to 6) packaged within a single viral envelope. The diameter of OBs was 0.9 to 1.3 μm with a mean of 1.152±0.116 μm. The complete sequence of the MaviMNPV polyhedrin (Polh) gene contained 735 nucleotides (GenBank accession number DQ399596). Phylogenetic analyses using the complete sequence of the Polh gene of MaviMNPV indicated that this virus clusters with Group I NPVs. The genome size of MaviMNPV estimated with restriction enzymes viz., HindIII, EcoRI, BglII and PstI was 113.41 ± 1.50 kbp. First instar LPB larvae were the most susceptible stage (LC₅₀ 2.053 × 10² OBs/ml) followed by second, third and fourth instars with the median lethal concentrations (LC₅₀s) 1.410 × 10³, 2.390 × 10³ and 2.636 × 10³ OBs/ml, respectively. This is the first record of this virus from this region. The first and second authors have equal contributions in this paper     

Insecticidal action of Quinomycin A from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. KN-0647, isolated from a forest soil

An actinomycete strain KN-0647 was isolated from a forest soil sample collected from Dali Cangshan mountain, Yunnan Province, China. The strain was identified as Streptomyces sp. according to the morphological, physiological characteristics and whole nucleotide sequence analysis of 16S rRNA gene, and could not be identified up to species level, just suggesting a potential new taxon. The ethyl acetate extract from this strain displayed growth inhibition on the test pathogenetic insects, such as Spodoptera exigua, Dendrolimus punctatus, Plutella xylostella, Aphis glycines and Culex pipiens. The active compound was isolated and identified through a combination of spectral and chemical methods (UR, MS, and ¹HNMR) as quinomycin A. This is the first report on the insecticidal activity of antibiotic quinomycin A.     

New cell lines derived from the black cutworm, Agrotis ipsilon, that support replication of the A. ipsilon multiple nucleopolyhedrovirus and several group I nucleopolyhedroviruses

New cell lines were recently developed from the embryos of the black cutworm, Agrotis ipsilon (Lepidoptera: Noctuidae). A primary culture was initiated from 4-day-old A. ipsilon eggs in ExCell420 medium supplemented with 5% fetal bovine serum. This initial culture produced sufficient cell growth to allow subcultivation and eventually led to the establishment of eight distinct strains. Two of these strains (AiE1611T and AiEd6T) were selected for further characterization. Extracts of these strains were compared to an extract from A. ipsilon eggs by isozyme analysis and shown to be from the same species. Both strains were susceptible to infection by the A. ipsilon multiple nucleopolyhedrovirus (AgipMNPV), as well as to lepidopteran group I NPVs from A. californica, Anagrapha falcifera, Anticarsia gemmatalis, Galleria mellonella, Helicoverpa armigera, Plutella xylostella, and Rachiplusia ou, with large numbers of occlusion bodies produced in most of the inoculated cells. The cell lines did not support the replication of group II NPVs from Helicoverpa zea, Lymantria dispar, and Spodoptera exigua. Both cell lines produced confluent monolayers in plaque assays and supported the formation of plaques upon infection with AgipMNPV and Autographa californica (Ac)MNPV. Twenty AgipMNPV plaques were picked from either AiE1611T or AiEd6T monolayers, and the plaque isolates were serially passaged three times through A. ipsilon cells. Only one isolate from AiE1611T cells exhibited genotypic variation in the form of an altered restriction fragment profile. Our results suggest these new lines can be useful in the study of AgipMNPV and A. ipsilon cellular and molecular biology.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.