Barley-based medium for the cost-effective production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Summary Studies conducted for the multiplication of Bacillus thuringiensis (Bt) using barley Hordeum vulgare as the carbon source led to the development of a protocol for the cost-effective, mass production of Bt. The production employs the simple shake flask method and can be easily adopted with a production potential of 1.5 kg Bt per day approximately at an overall production cost of Rs. 360 kg^-1 (8 US dollars). The protocol is suitable for promoting localized production of Bt at the village/district level. The product when tested as 0.1% (w/v) spray against the castor semilooper, Achoea janata proved highly effective, causing immediate feeding cessation of the larvae followed by 85% and 100% mortality by 48 and 72 h after treatment, respectively.     

Expression of the cry1EC gene in castor (Ricinus communis L.) confers field resistance to tobacco caterpillar (Spodoptera litura Fabr) and castor semilooper (Achoea janata L.)

Castor (cv. DCS-9) has been transformed through Agrobacterium-mediated and particle gun bombardment methods using appropriate vectors containing the Bt chimeric gene cry1EC driven by enhanced 35S promoter. About 81 and 12 putative transformants were regenerated following selection on hygromycin and kanamycin, respectively. Southern analysis of DNA extracted from T₀ plants confirmed integration of the introduced gene in castor genome. The integration and inheritance of the introduced genes was demonstrated up to T₄ generation by PCR and Southern analysis. Southern analysis of two events having single and two copies showed the same pattern of integration in the subsequent generations. Insect feeding experiments conducted in the laboratory by releasing neonate larvae of castor semilooper and S. litura on leaf tissues excised from transgenic and control plants showed varying degrees of larval mortality and slow growth in larvae fed on transgenic leaf tissue. Field bioassays against Spodoptera litura and castor semilooper conducted for eight events in T₁–T₄ generations under net confinement were more informative and events conferring resistance to the two major defoliators were identified.     

Phagocytic activity and encapsulation rate of Galleria mellonella larval haemocytes during bacterial infection by Bacillus thuringiensis

The bacterium Bacillus thuringiensis (Bt) is a pathogen of many insect species and is actively used in biocontrol. After the peroral inoculation of Galleria mellonella by the Bt in 5% sublethal concentration (LC₅), a 1.5-fold increase in the phagocytic activity of infected larvae has been registered on the second and third days after the inoculation. With the increase of Bt-inoculum amount to 15% of sublethal concentration (LC₁₅), a further increase of the phagocytic activity and enhanced encapsulation rates in the haemolymph of infected larvae has been observed. The enhanced cellular immunity during the bacteriosis seems to have resulted from the destruction of midgut epithelium cells followed by the subsequent exposure of gut content to lymph factors activating the immune system of haemocoel.     

Patch-clamp study of the apical membrane of the midgut of Manduca sexta larvae: direct demonstration of endogenous channels and effect of a Bacillus thuringiensis toxin

The patch-clamp technique was applied to the apical membrane of epithelial midgut cells of a lepidoptera, Manduca sexta L. Access to the apical membrane, the main target site of Bacillus thuringiensis (Bt) toxins, was achieved by using freshly isolated larval midgut preparations mounted onto holding glass pipettes. The epithelial cells retained their functional integrity, as evidenced by the magnitude of intracellular potentials recorded with microelectrodes. With standard 32 mM K(+) solution in the bath and the patch-clamp pipette, endogenous channel activity was detected in about 50% of experiments, mainly in moulting larvae and larvae that had been kept at reduced temperature for at least two days prior to the experiments. In both cell-attached and inside-out patch-clamp configurations, different types of channel were observed, with conductances varying between about 5 and 50 pS and different conducting properties. Addition of trypsin-activated Cry1Ac Bt toxin in the patch-clamp pipette triggered, after a delay, large conductances of a few nanosiemens. This is the first study allowing exploration, in the intact midgut, of the properties of apical membrane channels and the direct interaction between the apical membrane of epithelial cells and pathogenic agents such as Bt toxins.     

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC₅₀) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and −0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.     

Effects of Bt plants on the development and survival of the parasitoid Cotesia plutellae (Hymenoptera: Braconidae) in susceptible and Bt-resistant larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

A range of crops have been transformed with delta-endotoxin genes from Bacillus thuringiensis (Bt) to produce transgenic plants with high levels of resistance to lepidopteran pests. Parasitoids are important natural enemies of lepidopteran larvae and the effects of Bt plants on these non-target insects have to be investigated to avoid unnecessary disruption of biological control. This study investigated the effects of Cry1Ac-expressing transgenic oilseed rape (Brassica napus) on the solitary braconid endoparasitoid Cotesia plutellae in small-scale laboratory experiments. C. plutellae is an important natural enemy of the diamondback moth (Plutella xylostella), the most important pest of brassica crops world-wide. Bt oilseed rape caused 100% mortality of a Bt-susceptible P. xylostella strain but no mortality of the Bt-resistant P. xylostella strain NO-QA. C. plutellae eggs laid in Bt-susceptible hosts feeding on Bt leaves hatched but premature host mortality did not allow C. plutellae larvae to complete their development. In contrast, C. plutellae developed to maturity in Bt-resistant hosts fed on Bt oilseed rape leaves and there was no effect of Bt plants on percentage parasitism, time to emergence from hosts, time to adult emergence and percentage adult emergence from cocoons. Weights of female progeny after development in Bt-resistant hosts did not differ between plant types but male progeny was significantly heavier on wildtype plants in one of two experiments. The proportion of female progeny was significantly higher on Bt plants in the first experiment with Bt-resistant hosts but this effect was not observed again when the experiment was repeated.     

Infection by the microsporidium Vairimorpha necatrix (Microspora: Microsporidia) elevates juvenile hormone titres in larvae of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae)

The effects of infection by a microsporidium, Vairimorpha necatrix (Kramer), on the endogenous levels of juvenile hormones in tomato moth (Lacanobia oleracea L.) larvae were investigated. Levels of juvenile hormone II (JH II) were 10-fold greater in the infected larvae on day two of the sixth stadium but no significant difference was observed on day seven. Juvenile hormone I (JH I) was also detected in day two and day seven sixth instar infected larvae but was not detected in non-infected larvae. The duration of the fifth and sixth stadia was significantly longer for infected larvae when compared with non-infected larvae. No evidence was found to suggest that supernumerary moults are a feature of infection by V. necatrix in L. oleracea larvae. Experiments were performed to determine whether the elevation in JH levels, which probably prevents pupation, is an adaptive mechanism of the microsporidium for extending the growth phase of the host, thereby allowing increased spore production. A proportion of infected larvae were collected on days 9 and 24 of the sixth stadium and spore extracts prepared from each larva. These days represent the average duration of the sixth stadium required for uninfected larvae to reach pupation, and the average number of days that V. necatrix-infected larvae survive in the sixth stadium before dying from infection. The mean spore yields from infected larvae 24 days into the sixth stadium were significantly higher than the spore yields obtained from day nine sixth instar larvae. The hypothesis that V. necatrix manipulates host endocrinology (i.e. prolong the host larval state to maximise spore yield) is discussed in context with the results obtained.     

The effect of temperature and duration of exposure of potato tuber moth (Lepidoptera: Gelechiidae) in infested tubers to the biofumigant fungus Muscodor albus

The endophytic fungus, Muscodor albus produces several volatile compounds (alcohols, esters, ketones, acids and lipids) that are biocidal for a range of organisms including plant pathogenic bacteria and fungi, nematodes and insects. We studied the effects of these volatiles on 3-day-old potato tuber moth larvae within infested tubers inside sealed chambers. The length of exposure to M. albus significantly affected mortality of larvae, calculated as percentage of larvae failing to survive to the adult stage. Exposure durations of 3, 7, or 14 days at 24 degrees C followed by incubation in fresh air at 27 degrees C until emergence resulted in mortalities of 84.2, 95.5 and 99.6%, respectively. However, the longer exposures also resulted in increased levels of carbon dioxide (CO(2)) that are unacceptable for tuber storage. Effects of M. albus on larval survival was also monitored at 10, 15 and 24 degrees C, using an exposure duration of 7 days followed by incubation in clean air at 27 degrees C until emergence. Mortality of larvae was sharply reduced at the lower temperatures resulting in 50.8, 76.8, and 95.4% mortality, respectively. Tuber storage conditions, especially cooling rates, are discussed with respect to using M. albus as a fumigant without simultaneously producing unacceptable (for tuber storage) levels of CO(2).     

Incubation period, spore egestion and horizontal transmission of Nosema fumiferanae (Microsporidia: Nosematidae) in spruce budworm (Choristoneura sp., Lepidoptera: Tortricidae): the role of temperature and dose

Various instars of Choristoneura occidentalis were fed with a range of doses of Nosema fumiferanae and reared at 20, 24 and 28 degrees C to determine the influence of temperature and dose on the time to spore egestion and the number of spores egested in the frass. When larvae were fed in the third stadium, as few as 10(2) spores per larva initiated infection, and both onset of spore egestion and the number of spores egested were affected by a complex relationship between temperature and inoculation dose. Onset of spore egestion varied from 11 to 15 days postinoculation. At 20 degrees C, the onset was delayed and spore production decreased with increasing inoculation dose whereas at higher temperatures spores were first egested at the lowest dose and spore production increased with dose. When larvae were fed spores in the fifth and sixth stadium, no spores were egested because pupation occurred before completion of the incubation period. To assess the effect of temperature on horizontal transmission, Choristoneura fumiferana larvae fed with 10(4) N. fumiferanae spores per larva were reared with uninfected larvae at 15, 20 and 25 degrees C. At 15 degrees C, we observed the highest degree of horizontal transmission, defined by the largest change in N. fumiferanae prevalence, even though the density of spores available for horizontal transmission was the lowest. Infected adults eclosed later than uninfected adults and the time to eclosion was also dependent on sex and temperature. We relate our experimental findings to consequences for horizontal and vertical transmission of N. fumiferanae in spruce budworm populations.     

Starvation induced stress and the susceptibility of the Colorado potato beetle,Leptinotarsa decemlineata,to infection by Beauveria bassiana

Starvation of second instar Colorado potato beetle larvae for 24h immediately after treatment with Beauveria bassiana conidia increased susceptibility to the pathogen and subsequent sporulation of cadavers but decreased time to larval death. In feeding studies, B. bassiana-treatment had no effect on subsequent larval development, and mortality occurred 5-6 days after treatment. Twenty-four hours of starvation alone retarded subsequent larval development but did not affect mortality. Mortality of B. bassiana-treated starvation stressed larvae occurred 4-5 days after treatment. Both B. bassiana treatment and 24h starvation significantly reduced total foliage consumption and daily weight gains. On the day of treatment, B. bassiana had no effect on the efficiency with which food was converted to biomass (ECI). ECI was not affected by B. bassiana or starvation alone on the day following treatment but was significantly affected by a combination of both. When larvae were exposed to a range of limited food quantities, ECI decreased with decreasing food availability but only extreme stress (starvation for 24h) increased susceptibility to B. bassiana. Topical application of Dacryodes excelsa resin (an antifeedant) to potato leaves caused a concentration dependent reduction in foliage consumption and weight gain by second instar larvae but did not affect larval mortality. When larvae were exposed to a fixed concentration of B. bassiana and a range of antifeedant concentrations there were significant linear relationships between 24h larval weight gain and mortality and 24h larval weight gain and sporulation. The interaction between starvation stress and the susceptibility to B. bassiana infection is discussed and its possible implications in pest management considered.     

Low salinity stress experienced by larvae does not affect post-metamorphic growth or survival in three calyptraeid gastropods

Marine larvae that experience some sub-lethal stresses can show effects from those stresses after metamorphosis, even when they seem to recover from those stresses before metamorphosis. In this study we investigated the short and long-term effects of exposing the larvae of three calyptraeid gastropods (Crepidula fornicata, Crepidula onyx, and Crepipatella fecunda) to temporary reductions in salinity. Larvae of all three species showed slower larval growth rates, longer time to metamorphic competence, and substantial mortality after being stressed in seawater at salinities of 10, 15, and 20 for less than 48 h. Larval tolerance to low salinities varied widely within and among species, but longer stresses at lower salinities were generally more harmful to larvae. However, larvae in nearly all experiments that were able to metamorphose survived and grew normally as juveniles; there were no documented “latent effects.” For all three species, starving larvae in full-strength seawater was not as harmful as exposing larvae to low salinity stress, indicating that detrimental effects on larvae were caused by the salinity stress per se, rather than by an indirect effect of salinity stress on feeding. C. fornicata that were stressed with low salinity as juveniles were more tolerant of the stress than larvae: all stressed juveniles lived and showed reduced growth rates for no more than 3 days. Our data suggest that even though reduced salinity is clearly stressful to the larvae of these 3 gastropod species, metamorphosis seems to generally provide individuals with a fresh start.     

The compatibility of a nucleopolyhedrosis virus control with resistance management for Bacillus thuringiensis: co-infection and cross-resistance studies with the diamondback moth, Plutella xylostella

The use of genetically modified crops expressing Bacillus thuringiensis (Bt) toxins can lead to the reduction in application of broad-spectrum pesticides and an increased opportunity for supplementary biological control. Bt microbial sprays are also used by organic growers or as part of integrated pest management programs that rely on the use of natural enemies. In both applications the evolution of resistance to Bt toxins is a potential problem. Natural enemies (pathogens or insects) acting in combination with toxins can accelerate or decelerate the evolution of resistance to Bt. In the present study we investigated whether the use of a nucleopolyhedrovirus (AcMNPV) could potentially affect the evolution of resistance to the Bt toxin Cry1Ac in Plutella xylostella. At low toxin doses there was evidence for antagonistic interactions between AcMNPV and Cry1Ac resistant and susceptible insects. However, this antagonism was much stronger and more widespread for susceptible larvae; interactions were generally not distinguishable from additive for resistant larvae. Selection for resistance to Cry1Ac in two populations of P. xylostella with differing resistance mechanisms did not produce any correlated changes in resistance to AcMNPV. Stronger antagonistic interactions between Bt and AcMNPV on susceptible rather than resistant larvae can decrease the relative fitness between Bt-resistant and susceptible larvae. These interactions and the lack of cross-resistance between virus and toxin suggest that the use of NPV is compatible with resistance management to Bt products.     

Extended in vitro culture of Microplitis croceipes teratocytes and secretion of TSP14 protein

Teratocytes, cells which originate from the serosal membrane of some Braconidae and Scelionidae, can be found in the hemocoel of permissive hosts during part or all of the developmental time of the parasitoid larva. Teratocytes from Microplitis croceipes are known to secrete biologically active proteins, which contribute to developmental arrest and failure to pupate of Heliothis virescens larvae. One such protein, which has a molecular weight of approximately 14 kDa is called TSP14. The presence of parasitoid larvae is essential to maintain teratocytes under in vitro conditions with protein-free EX-CELL 400. The teratocyte viability was maintained in vitro for at least 12 days in the presence of larvae when medium was exchanged every three days. Western blots show that TSP14 was secreted during the entire period of exchanges. In the absence of parasitoid larvae, teratocyte viability was only 30% by day 6 and no TSP14 had been secreted. In the absence of parasitoid larvae, teratocytes maintained in vitro in EX-CELL 400 medium supplemented with 10% FBS remained viable for at least nine days and secreted TSP14 for at least six days. This suggests that parasitoid larval secretions are sufficient but not uniquely essential to maintain teratocyte viability. Parasitoid larvae maintained in the absence of teratocytes did not secrete TSP14 and their secretory products did not inhibit pupation of H. virescens larvae.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.     

Setting tools for the early assessment of the quality of thawed Pacific oyster (Crassostrea gigas) D-larvae

Parameters used to assess the survival of larvae after cryopreservation generally misestimate the damages that prevent larval development. The objectives of the present study were to 1) define the reliability of the survival rate, assessed at 2 and 7 days post fertilization, to estimate Pacific oyster larval quality after thawing, and 2) select complementary tools allowing an early and reliable estimation of their quality. Oyster larvae were reared for 25 h after fertilization at 19 °C and cryopreserved at early D-stage. Then, thawed larvae were incubated in 2-L beakers. At 2 days post fertilization, the survival rate of thawed Pacific oyster larvae was lower than that of fresh larvae for only one experiment (Experiment 3) among the four identical experiments carried out in this work (Experiments 1-4). By contrast, the survival of thawed larvae, as assessed 7 days after fertilization, was lower than that of fresh larvae for the four experiments. These results confirm that the quality of thawed larvae is lower than that of fresh larvae and that the survival rate, estimated 2 days post fertilization, is not adapted to a reliable estimation of the subsequent development ability of thawed larvae. Then, complementary parameters were tested at 2 days: the movement characteristics (Experiments 1 and 2) and the morphologic features (Experiments 3 and 4) of thawed larvae. Compared to values observed on fresh larvae, the percentage of thawed motile larvae was different for only one experiment (Experiment 2) of the two. Compared to control, a reduced Average Path Velocity (VAP) of larvae (determined at the D-larval stage using a CASA-Computer Assisted Sperm Analysis-system) was observed after thawing for both experiments (Experiments 1 and 2), suggesting the ability of larval movement velocity to assess the decrease of the quality of thawed oyster larvae. Using an ASMA (Automated Sperm Morphology Analysis) device, a lower area of thawed larvae was observed, compared to control and for the two experiments (Experiments 3 and 4). By contrast, the Crofton perimeter of thawed larvae was lower than that of control larvae for only one experiment (Experiment 3) and no significant difference of circularity between fresh and thawed larvae was recorded for Experiments 3 and 4. In conclusion, changes in the movement velocity (assessed by CASA) and in the area (measured by ASMA) of D-larvae allow an early and reliable estimation of the quality of thawed Pacific oyster larvae.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Microsporidian infections in Lymantria dispar larvae: interactions and effects of multiple species infections on pathogen horizontal transmission

The interactions in multiple species infections and effects on the horizontal transmission of three microsporidian species, Vairimorpha disparis, Nosema lymantriae and Endoreticulatus schubergi, infecting Lymantria dispar were evaluated in the laboratory. Simultaneous and sequential inoculations of host larvae were performed and the resulting infections were evaluated. Test larvae were exposed to the inoculated larvae to measure horizontal transmission. Dual species infections demonstrated interspecific competition between Nosema and Vairimorpha in the host larvae, but no observable competition occurred between Endoreticulatus and either of the other microsporidian species. Timing of inoculation was an important factor determining the outcome of competition between Nosema and Vairimorpha. The species inoculated first showed a higher rate of successful establishment; a time lag of 7 days between inoculations allowed the first species to essentially exclude the second. The microsporidia differed in efficiency of horizontal transmission. Nosema and Endoreticulatus were transmitted at very high rates, close to 100%. Horizontal transmission of Vairimorpha was less efficient, ranging from 25% to a maximum of 75%. The patterns of infection observed in inoculated larvae were reflected in the test larvae that acquired infections in the horizontal transmission experiments. Competition with Vairimorpha suppressed horizontal transmission of Nosema after simultaneous and sequential inoculation. In simultaneous inoculation experiments Endoreticulatus had no effect on transmission of Nosema and Vairimorpha.