Spodoptera Exigua Larvae Research Articles

Context‐dependent defence in terrestrial plants: the effects of light and nutrient availability on plant resistance against herbivory

AbstractThe context‐dependent defence (CDD) hypothesis predicts that defence levels of plant species against herbivory are not fixed but vary with environmental conditions, in a way that is specific for plant species that share evolutionary adaptations to resource conditions exemplified by similar maximum relative growth rates. More specifically, we expected plants from resource‐poor environments to display high defence levels but not when grown under resource‐rich conditions, whereas the reverse – plants from resource‐rich conditions displaying low defence levels but not when grown under resource‐poor conditions – is not necessarily the case. In this study, we used multiple‐choice bioassays in which leaf discs were fed to larvae of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) as an efficient and effective way of indicating plant defence levels. This generalist herbivore was capable of detecting both inter‐ and intraspecific differences in defence among plant species. The CDD was tested by exploring the effects of various experimental resource conditions (light, nutrients) upon the herbivore preferences and by comparing these preferences with the maximum relative growth rate of plant species. The experimental results provide general support for the CDD hypothesis with respect to nutrient‐level variation but the effects were not related to the origin of the plant species tested. Variation in light conditions did not result in consistent effects upon herbivore preferences. The CDD therefore can be formulated more precisely as: defence levels of plant species vary under different environmental conditions but in a way that is specific for plant species that share evolutionary adaptations to similar nutrient conditions. This more precise CDD hypothesis is a useful addition to existing optimal‐defence theory because of its focus on the possible plastic effects of resource conditions upon plant defence levels. This is relevant when designing experimental plant–herbivore studies.

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

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

Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine-Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry(-)B. The recombinant plasmid was stably maintained over 240 generations in Cry(-)B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l(-1) Na(2)CO(3)-0.2% beta-mercaptoethanol buffer at a wide range of alkaline pH values, and what's more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.

Analysis of promoter activity of selected Cotesia plutellae bracovirus genes

In a previous study, we cloned 27 discrete genome segments of Cotesia plutellae bracovirus (CpBV) and provided the complete nucleotide sequences and annotation. Seven putative coding regions were predicted from one of the largest segments, CpBV-S30. The activity of promoters associated with six predicted ORFs from this segment were investigated using both transient and baculovirus expression assays with enhanced green fluorescent protein as a reporter gene. CpBV promoters showed activity earlier than the polyhedrin promoter and the activity of some of these promoters was superior to that of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ie-1 promoter in the baculovirus expression assays. The promoter of ORF3004 showed the highest level of activity in insect cells, exhibiting 24 % of the activity obtained with the AcMNPV polyhedrin promoter in Sf9 cells. In Spodoptera exigua larvae, the ORF3006 promoter showed the highest activity, with about 35 % of the activity measured with the polyhedrin promoter. In addition, analysis of the ORF3006 promoter revealed that the region between -382 and -422 from the translation start point was critical for activity of this promoter. These results suggest that the CpBV-S30 promoters characterized here could be useful tools in a variety of biotechnological applications, such as gene expression analyses and insecticide development.

Broad‐spectrum cross‐resistance in Spodoptera exigua from selection with a marginally toxic Cry protein

Spodoptera exigua (Hübner) has developed resistance to a wide range of chemical insecticides. Products based on Bacillus thuringiensis Cry toxins are used in integrated pest management as an ecologically friendly alternative for pest control. Since there are few B. thuringiensis Cry proteins highly active against S. exigua, it is desirable to apply appropriate resistance management strategies to prevent the evolution of resistance to these proteins. Spodoptera exigua larvae were selected with Cry1Ab, a protein with low activity against this pest. Selected larvae developed > 30-fold resistance to Cry1Ab in 13 generations, relative to an unselected strain. The estimated realised heritability (h(2)) for the first five generations of selection was 0.15. Cross-resistance was also observed to the more active proteins Cry1Ca, Cry1Da and Cry1Fa (>20, 26 and > 8 respectively). The activity of midgut proteases to degrade the ingested toxin was tested, although no differences in activity were found between selected and unselected larvae. Spodoptera exigua is able to evolve cross-resistance to highly active Cry proteins when exposed to a protein with marginal toxicity to this species. It is important to take this into account in areas where S. exigua is a secondary pest and B. thuringiensis Cry1A toxins are used to control other pests.

Identification of an Entomopathogenic Bacterium, Serratia sp. ANU101, and Its Hemolytic Activity

Four different bacterial colonies were isolated from an old stock of an entomopathogenic nematode, Steinernema monticolum. They all showed entomopathogenicity to final instar larvae of beet armyworm, Spodoptera exigua, by hemocoelic injection. However, they varied in colony form, susceptibility to antibiotics, and postmortem change of the infected host insects. Biolog microbial identification and 16S rDNA sequence analyses indicate that these are four different species classified into different bacterial genera. owing to high entomopathogenicity and a cadaver color of infected insect host, Serratia sp. was selected as a main symbiotic bacterial species and analyzed for its pathogenicity. Although no virulence of Serratia sp. was detected at oral administration, the bacteria gave significant synergistic pathogenicity to fifth instar S. exigua when it was treated along with a spore-forming entomopathogenic bacterium, Bacillus thuringiensis. The synergistic effect was explained by an immunosuppressive effect of Serratia sp. by its high cytotoxic effect on hemocytes of S. exigua, because Serratia sp. caused septicemia of S. exigua when the bacterial cells were injected into S. exigua hemocoel. The cytotoxic factor(s) was present in the culture medium because the sterilized culture broth possessed high potency in the cytotoxicity, which was specific to granular cells and plasmatocytes, two main immune-associated hemocytes in insects.

Solubilization, Activation, and Insecticidal Activity ofBacillus thuringiensisSerovar thompsoni HD542 Crystal Proteins

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

Insecticidal activities of recombinant Autographa californica nucleopolyhedrovirus containing a scorpion neurotoxin gene using promoters from Cotesia plutellae bracovirus

To investigate the usefulness of early-expressed promoters from Cotesia plutellae bracovirus (CpBV) in improvement of insecticidal activities of baculovirus, recombinant AcNPVs, Ac3003ProAaIT, Ac3004ProAaIT, Ac3005ProAaIT and Ac3006ProAaIT expressing scorpion neurotoxin, AaIT, under the control of ORF3003, ORF3004, ORF3005 and ORF3006 promoter, respectively were constructed. Among these recombinant viruses, Ac3006ProAaIT showed highest insecticidal activity against 3rd instar larvae of Spodoptera exigua. These results suggested that early promoters from CpBV could be successfully applied to improve pathogenicity of baculoviruses.

Spore stage expression of a vegetative insecticidal gene increase toxicity of Bacillus thuringiensis subsp. aizawai SP41 against Spodoptera exigua

To enhance the toxicity of the Bacillus thuringiensis subsp. aizawai strain SP41 (SP41), the vegetative insecticidal protein (Vip) gene vip3A from SP41 was redirected to the sporulation stage by replacing its native promoter with the strong promoter P19 of the cry11Aa operon. Compared to the wild type, SP41 with PVIP ( vip3A with its native promoter and ter) had the relative expression ratios of 457, 548, and 290 at 8, 14, and 20 h of cultivation, respectively, as measured by quantitative reverse transcription polymerase chain reaction (PCR). SP41 transformed by P19VIP ( vip3A controlled by P19 promoter with vip3A ter) showed higher expressions (23, 2055, 1831) at the same time points. SP41 with P19VIP20 ( vip3A controlled by P19 promoter and containing P20 and operon ter) had the lowest expression levels (3, 11, 9) at any time point. SDS-PAGE analysis of proteins in the culture supernatant of the P19VIP at 8, 14, and 20 h demonstrated a significant increase in Vip3A at the sporulation stage. Using the surface contamination bioassay, the 50% lethal concentration (LC 50) of whole culture of PVIP, P19VIP, and P19VIP20 at 20 and 48 h of cultivation against Spodoptera exigua larvae were (68.3, 21.2, and 60.2 μg cm −2) and (69.8, 41.8, and 74.6 μg cm −2), respectively, compared with 86.6 and 104.4 μg cm −2 for SP41. The results showed that Vip from P19VIP, expressed at spore stage at 20 and 48 h, can increase the toxicity of SP41 for 4.1- and 2.5-fold, respectively.

Starvation Effects on Larval Development of Beet Armyworm (Lepidoptera: Noctuidae)

Sporadic starvation of herbivorous insect pests, such as the beet armyworm Spodoptera exigua (Hübner), often occurs due to host plant scarcity and dislodgement from host plants resulting from inclement weather, enemy attack, and other perturbations. We hypothesized that (1) starvation of beet armyworm in earlier instars would increase the possibility of undergoing supernumerary instars greater than would be the case for later instars; and (2) starvation of beet armyworm in earlier instars would prolong developmental time more than starvation in later instars. We tested these hypotheses by starving beet armyworm larvae for 48 h in the first, third, and fifth instars, and monitoring their development. Beet armyworms normally have 5 instars before pupation. Significantly more larvae underwent 6 larval instars (20.22 ± 4.35%) when starved for 2 d in the first instar compared with those starved for 2 d in the third (2.0 ± 2.00) and fifth instars (0.0), and relative to unstarved larvae (0.0). The number of instars that S. exigua larvae will experience can be predicted as early as the third instar-head capsule widths in this instar clearly differentiate for those destined to experience 5 and 6 instars. Developmental times of starved larvae also were prolonged compared with unstarved larvae. Developmental times (mean ± SE in days) of larvae starved in the first instar (20.32 ±0.16) were significantly longer than those starved in the third (17.04 ± 0.20) or fifth instars (16.92 ± 0.10), or than unstarved larvae (14.62 ± 0.25). Timing of starvation significantly affected the pupal weight of larvae undergoing 5 instars. This suggests that (1) starvation of 2 d in very early instars (the first instar) increases the pupal weight at the cost of prolonged developmental time; and (2) starvation of 2 d in later instars (the fifth instar) leads to lower pupal biomass, presumably because of inability to compensate for the loss.

Evaluations of Bollgard®, Bollgard II®, and WideStrike® Technologies against Beet and Fall Armyworm Larvae (Lepidoptera: Noctuidae)

Transgenic cottons containing the Bollgard®, Bollgard II® and WideStrike® traits were grown in 2005 and 2007 to examine the efficacy against beet armyworm Spodoptera exigua (Hübner) and fall armyworms S. frugiperda (J. E. Smith). Results suggest that both dual-gene traits are more efficacious against these armyworm species than Bollgard®. In these studies, WideStrike® appears to be more efficacious against fall armyworms than Bollgard II®, while Bollgard II® is more efficacious against beet armyworms than WideStrike®. Possible reasons for these differences in efficacy are discussed.

Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling.

Plant volatiles emitted by Medicago truncatula in response to feeding larvae of Spodoptera exigua are composed of a complex blend of terpenoids. The cDNAs of three terpene synthases (TPSs), which contribute to the blend of terpenoids, were cloned from M. truncatula. Their functional characterization proved MtTPS1 to be a β-caryophyllene synthase and MtTPS5 to be a multi-product sesquiterpene synthase. MtTPS3 encodes a bifunctional enzyme producing (E)-nerolidol and geranyllinalool (precursors of C11 and C16 homoterpenes) from different prenyl diphosphates serving as substrates. The addition of jasmonic acid (JA) induced expression of the TPS genes, but terpenoid emission was higher from plants treated with JA and the ethylene precursor 1-amino-cyclopropyl-1-carboxylic acid. Compared to infested wild-type M. truncatula plants, lower amounts of various sesquiterpenes and a C11–homoterpene were released from an ethylene-insensitive mutant skl. This difference coincided with lower transcript levels of MtTPS5 and of 1-deoxy-d-xylulose-5-phosphate synthase (MtDXS2) in the damaged skl leaves. Moreover, ethephon, an ethylene-releasing compound, modified the extent and mode of the herbivore-stimulated Ca2+ variations in the cytoplasm that is necessary for both JA and terpene biosynthesis. Thus, ethylene contributes to the herbivory-induced terpenoid biosynthesis at least twice: by modulating both early signaling events such as cytoplasmic Ca2+-influx and the downstream JA-dependent biosynthesis of terpenoids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-007-0631-y) contains supplementary material, which is available to authorized users.

Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene

To search for novel Vip3A proteins for controlling insect pests. A pair of universal primers was designed based on the conserved regions of five vip3A genes. Amplified products were digested with the HindIII and EcoR enzymes so as to confirm different restriction fragment length polymorphism (RFLP) patterns used to identify vip3A-type genes. The vip3A gene types of 606 Bacillus thuringiensis strains were screened and three patterns of RFLP were successfully identified. Two novel vip3A genes were found and one of these, vip3Aa19, was further characterized and its product was confirmed toxic to Spodoptera exigua, Helicoverpa armigera and Plutella xylostella larvae. Partial sequences of another novel vip3A-type gene were obtained that shared 83% homology with that of the vip3Af1 gene. A polymerase chain reaction (PCR)-RFLP system we developed could be used for identifying novel vip3A-genes from B. thuringiensis strains. A novel Vip3A protein was found to have a broader insecticidal spectrum. The reported method is a powerful tool to find novel Vip3A proteins from large-scale B. thuringiensis strains. The novel Vip3A protein may be used to control insect pests or resistant insect pests by constructing genetically engineered strains or transgenic plants.

Increased SA in NPR1‐silenced plants antagonizes JA and JA‐dependent direct and indirect defenses in herbivore‐attacked Nicotiana attenuata in nature

The phytohormone jasmonic acid (JA) is known to mediate herbivore resistance, while salicylic acid (SA) and non-expressor of PR-1 (NPR1) mediate pathogen resistance in many plants. Herbivore attack on Nicotiana attenuata elicits increases in JA and JA-mediated defenses, but also increases SA levels and Na-NPR1 transcripts from the plant's single genomic copy. SA treatment of wild-type plants increases Na-NPR1 and Na-PR1 transcripts. Plants silenced in NPR1 accumulation by RNAi (ir-npr1) are highly susceptible to herbivore and pathogen attack when planted in their native habitat in Utah. They are also impaired in their ability to attract Geocorus pallens predators, due to their decreased ability to release cis-alpha-bergamotene, a JA-elicited volatile 'alarm call'. In the glasshouse, Spodoptera exigua larvae grew better on ir-npr1 plants, which had low levels of JA, JA-isoleucine/leucine, lipoxygenase-3 (LOX3) transcripts and JA-elicited direct defense metabolites (nicotine, caffeoyl putrescine and rutin), but high levels of SA and isochorismate synthase (ICS) transcripts, suggesting de novo biosynthesis of SA. A microarray analysis revealed downregulation of many JA-elicited genes and upregulation of SA biosynthetic genes. JA treatment restored nicotine levels and resistance to S. exigua in ir-npr1 plants. We conclude that, during herbivore attack, NPR1 negatively regulates SA production, allowing the unfettered elicitation of JA-mediated defenses; when NPR1 is silenced, the elicited increases in SA production antagonize JA and JA-related defenses, making the plants susceptible to herbivores.

Exposure to carbaryl leads to ultrastructural changes and alters activity of antioxidant enzymes in Spodoptera exigua (Lepidoptera: Noctuidae)

Abstract. The toxic effect of carbamate insecticide carbaryl on Spodoptera exigua (Lepidoptera: Noctuidae) larvae was examined. Insects appeared to be highly sensitive to it; numerous malfunctions and malformations were observed. The first ultrastructural alterations, including biological membrane swelling, nuclear condensation, and disintegration, and abnormalities around the mitochondria were observed even before massive mortality was noted. Scanning electron microscopy revealed changes in cuticle. Carbaryl depleted the wet weight of fat body mass and changed (in most of the cases elevated) the activity of antioxidant enzymes. The majority of these malformations are rather non‐specific and are common among animals exposed to xenobiotics. However, they all suggest serious disruptions in metabolism, in addition to indicating that reactive oxygen species may play an important role in the toxic effect of carbaryl.

Apoptosis is induced in the haemolymph and fat body of Spodoptera exigua larvae upon oral inoculation with Spodoptera litura nucleopolyhedrovirus

Spodoptera exigua multinucleopolyhedrovirus (SeMNPV) and Spodoptera litura nucleopolyhedrovirus (SpltNPV) are genetically similar, but the larvae of S. exigua are not susceptible to SpltNPV. The aim of this study was to identify whether any process was inhibiting SpltNPV infection at some point. S. exigua larvae infected with a high concentration of wild-type SpltNPV by oral inoculation produced a fatal infection in second- or third-instar S. exigua, but the dead larvae did not undergo liquefaction; in contrast, fourth-instar infected larvae remained healthy. RT-PCR analysis of total RNA from infected second-instar larvae targeting immediate-early (ie-0), early (dnapol), late (chit) and very late (polh) genes suggested that SpltNPV initiated infection in the non-susceptible hosts. Total DNA extracted from the haemocytes of infected larvae showed DNA ladders characteristic of apoptosis. Sections of tissue from infected third-instar larvae of S. exigua at 96 h post-inoculation, stained with haematoxylin and eosin, revealed a highly disrupted morphology in the fat body. Apoptosis in fat body tissue was detected using terminal deoxynucleotidyltransferase-mediated fluorescein-dUTP nick end labelling (TUNEL) assays. In situ hybridization revealed the presence of viral DNA within the TUNEL-positive area, indicating viral infection in this tissue. These results suggest that apoptosis limits viral propagation by reducing the number of SpltNPV-infected haemocytes and fat body cells and inhibits disseminated viral infection.

Functional response ofPicromerus bidens: effects of host plant

Abstract: The influence of three solanaceous plants (tomato, sweet pepper and eggplant) on the functional response of the predatory bugPicromerus bidensto densities of fourth‐instar larvae of the beet armywormSpodoptera exiguawas assessed. Logistic regression indicated a type II functional response on all host plants. Over all prey densities,P. bidenskilled significantly fewer fourth instars ofS. exiguaon tomato than on sweet pepper or eggplant (1.96 ± 0.17 vs. 4.37 ± 0.19 and 3.90 ± 0.15 larvae per predator per 24 h respectively). A higher theoretical maximum predation rate was estimated on sweet pepper (11.1 prey larvae per day) and eggplant (7.4) than on tomato (5.4). The mean number of prey killed per day byP. bidensfemales ranged from 0.78 at a density of one prey on tomato to 8.45 at a density of 24 prey on sweet pepper. The data indicated that the estimates of handling time (Th) and attack rate (a) were highly affected by host plant. Based on asymptotic 95% confidence intervals, a lower attack coefficient was found on tomato (0.02 h−1) than on sweet pepper or eggplant (0.07 and 0.11 h−1respectively). On the other hand, handling times were significantly longer on tomato (4.42 h) and eggplant (3.23 h) than on sweet pepper (2.15 h). This laboratory study suggests that plant characteristics influence the ability ofP. bidensto respond to changes in prey density.

Juvenile hormone analogs greatly increase the production of a nucleopolyhedrovirus

Abstract 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 LC 50 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.

Kinetics of plasmid transfer among Bacillus cereus group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44-56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 x 10(-6) CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.

Synergistic Effect of Entomopathogenic Bacteria (<I>Xenorhabdus</I> sp. and <I>Photorhabdus temperata</I> ssp. <I>temperata</I>) on the Pathogenicity of <I>Bacillus thuringiensis</I> ssp. <I>aizawai</I> Against <I>Spodoptera exigua</I> (Lepidoptera: Noctuidae)

Xenorhabdus sp. (X. sp.) and Photorhabdus temperata subsp. temperata (Ptt) are the symbiotic bacteria of the entomopathogenic nematodes, Steinernema monticolum and Heterorhabditis megidis, respectively. When the nematodes enter target insects, the symbiotic bacteria are released into the insect hemocoel. The bacteria cause septicemia and kill the insect host. When ingested, the bacteria caused high mortality of third-instar larvae of Spodoptera exigua, but did not show such high pathogenicity to the fifth-instar larvae. The high mortalities in the third-instar larvae seem to be caused by antibiotic activity against Bacillus cereus, a gut symbiont required for optimal development of S. exigua. To increase their pathogenicity in the fifth instar, the bacteria should be delivered into the hemocoel. To this end, Bacillus thuringiensis aizawai (Bt) as a synergist was used to facilitate entry of the bacteria from the gut lumen into the hemocoel of S. exigua by its disruption of the insect gut epithelium. The bacterial mixture treatment was highly synergistic against the fifth-instar larvae of S. exigua. The synergistic effects were shown by the successful infection of X. sp. or Ptt in the insect hemocoel. This research shows a possibility that Xenorhabdus or Photorhabdus can be applied to kill S. exigua by oral treatment in a mixture with Bt.