Effect of different additives on the persistence and insecticidal activity of native strains of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The persistence and insecticidal activity of native strains of Bacillus thuringiensis was evaluated in formulations containing different additives such as arrow-root powder, carboxy methyl cellulose (CMC), gum acacia, non-food grade (NFG) starch, and soluble starch. Persistence of B. thuringiensis varied with different additives used in the formulations. Among the different additives used, NFG starch provided maximum protection to B. thuringiensis and native strain 42 showed maximum persistence (83%) which was higher than that obtained in commercial formulation. In commercial formulation, the persistence of B. thuringiensis was 47% only after 3 d of spray. The feeding trials conducted on second instar larvae of H. armigera using leaves sprayed with NFG starch formulation revealed 70% larval mortality while commercial formulation showed 50% mortality during the same period.     

Facultative monophagy as a consequence of prior feeding experience: behavioral and physiological specialization in Colias philodice larvae

Summary Although newly-emerged Colias philodice readily accept Medicago sativa, Melilotus alba, and Coronilla varia, fifth instar larvae reared on any single plant species display a highly significant induced feeding preference for their rearing host. Forced host-switching reveals that fifth instar relative growth rate (RGR) on M. sativa and M. alba is significantly reduced by prior feeding on either alternative host. Moreover, regardless of rearing diet, switching to a novel host during the fifth instar results in reduced RGR, relative consumption rate (RCR), efficiency of conversion of digested food (ECD), and pupal weight. These results support the hypothesis that induction of feeding preference is an adaptive response that predisposes larvae to feed on the plant species they are most capable of utilizing for growth.     

Responses of production and storage walnut pests to Bacillus thuringiensis insecticidal crystal protein fragments

Recent advances in genetic engineering have provided the opportunity to induce walnut plants to produce Bacillus thuringiensis Berliner insecticidal crystal protein fragments (ICPFs) for insect control. We studied the effects of two ICPFs CryIA(b) and CrylA(c) previously shown to be encoded by the cryIA(b) and cryIA(c) genes in the B. thuringiensis strains HD-1 and HD-73, respectively. The lethal effects on larvae of codling moth, Cydia pomonella (L.), navel orangeworm, Amyelois transitella (Walker), and the major postharvest pest Indianmeal moth, Plodia interpunctella (Hübner), were investigated. Both proteins were toxic to the three species tested. Indianmeal moth larvae were the most susceptible and navel orangeworm the least; CryIA(b) was generally more toxic to navel orangeworm. Similar relationships resulted when ICPFs were incorporated into the diet. Both ICPFs caused decreased rate of development of navel orangeworm. Effects on pupal weight occurred only at the highest concentration (100 ng/cm²). Neither ICPF affected frequency of mating or fecundity. In addition to the lethal effects, the extended development times observed could have considerable effects on the population dynamics of the navel orangeworm and possibly other species.     

Caste-specific modulation of juvenile hormone III content and ecdysteroid titer in postembryonic development of the stingless bee,Scaptotrigona postica depilis

Summary Juvenile hormone III content and ecdysteroid titer were analyzed for larval and pupal development of the stingless bee,Scaptotrigona postica depilis. Castespecific differences in juvenile hormone III content were detected at three developmental phases: at the transition from the fourth to the fifth larval stadium, in the spinning phase of the fifth larval stadium, and shortly after the imaginal moult. During the fifth larval stadium, juvenile hormone content closely reflects corpora allata activity. Juvenile hormone synthesis may thus be responsible for the elevated hormone titer in spinning-phase queen larvae, a phase of known sensitivity for induction of queen characters by exogenous juvenile hormone. For ecdysteroids, two phases of caste-specific differences were found: in the pre-pupal phase, and shortly after the imaginal moult. In both periods the titer in queens is distinctly higher compared to workers.Abbreviations Im imago 1 day after eclosion - L3, L4, L5 larval instars 3, 4, and 5 - L5F1, L5F2 substages of feeding phase in fifth larval instar - L5S1, L5S2, L5S3 substages of spinning phase in fifth larval instar - PP1, PP2 substages of prepupal phase - Pw white eyed pupa - Pp pink eyed pupa - Pr red eyed pupa - Pd dark eyed pupa - Pdl, Pdm, Pdd dark eyed pupa with progressive tanning of cuticle - RIA radioimmunoassay     

Effect of elevated atmospheric carbon dioxide on the use of foliar application of Bacillus thuringiensis

Toxins from Bacillus thuringiensis have beenused as pest management tools for more than 50 years. The effect of these toxins depends on the quantityof Bacillus thuringiensis (Bt) toxins ingestedby susceptible insects. Food ingestion is affected byCO₂ concentration; plants grown in elevatedCO₂ often have increased carbon/nitrogen ratios(C/N), resulting in greater leaf area consumption. Therefore, we hypothesized that elevated CO₂would improve the efficacy of foliar applications ofB. thuringiensis. Cotton plants were grown ateither ambient (360–380 l/l) or elevated CO₂(900 l/l). Groups of plants in both CO₂treatments were exposed to low (30 mg/kg soil/week) orhigh (130 mg/kg soil/week) nitrogen (N) fertilizationlevels in a split plot design. The resulting plantswere assessed for N and carbon (C) contents. Leafdisks from the same plants were dipped in a Btsolution and then fed to Spodoptera exigua(Hübner), an insect species of considerableeconomic importance. Elevated CO₂ significantlyreduced total N, and increased the C/N. Nitrogenfertilization significantly affected consumption byearly stadia larvae, larval weight gain, and relativegrowth rate (RGR). Interactions between CO₂concentration and N fertilization level significantlyimpacted late stadia larval food consumption, andthrough differential Bt toxin intake, affectedduration of larval stage and mortality to the adultstage. We conclude that the elevated atmosphericCO₂ concentrations expected in the next centurywill interact with commercial fertilization practicesto enhance the efficacy of B. thuringiensisformulations applied topically to crops. Theimplications for improved control are discussed.     

Strategy for orchard use of Bacillus thuringiensis while minimizing impact on Choristoneura rosaceana parasitoids

Trials were conducted to study how spring Bacillus thuringiensis Berliner subsp. kurstaki treatments on apple may be timed to maximize the survival of parasitoids of the obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae), found in the southern interior of British Columbia, Canada. Orchard collections verified that second through fourth instar obliquebanded leafrollers were found in varying proportions from pink through the petal fall stage of apple development when spring B. thuringiensis treatments are applied vs. lepidopteran pests. Laboratory‐reared second through fourth instar obliquebanded leafrollers, unparasitized and parasitized by one of three native parasitoid species, were fed untreated apple leaves or leaves treated with B. thuringiensis. The highest mortality of unparasitized obliquebanded leafrollers occurred when fourth instars were exposed to B. thuringiensis‐treated leaves; B. thuringiensis‐induced mortality in the unparasitized second and third instars was less than 50%. The consumption of B. thuringiensis‐treated leaves by host larvae significantly increased the percentage of dead host larvae in all parasitized and unparasitized treatments. However, because of the low susceptibility of this leafroller species to B. thuringiensis, relatively high numbers (38–43%) of three obliquebanded leafroller parasitoid species were able to survive the consumption of B. thuringiensis by second and third instar host larvae. Fourth instar obliquebanded leafrollers were found at the full bloom and petal fall stage of apple development in the orchard, at which time B. thuringiensis treatments are recommended for optimal leafroller control. The highest parasitoid mortality due to host mortality was recorded in Apophua simplicipes Cresson (Hymenoptera: Ichneumonidae) and Macrocentrus linearis (Nees) (Hymenoptera: Braconidae), when the hosts were treated as fourth instars. Both of these parasitoids emerge from fifth and sixth instar obliquebanded leafrollers. Bacillus thuringiensis did not have as negative an impact on Apanteles polychrosidis Viereck (Hymenoptera: Braconidae), which emerges when the host is in the fourth instar. When leafroller mortality and parasitism were combined, the B. thuringiensis treatment did not significantly increase host elimination above that of parasitism alone, except for larvae parasitized by A. simplicipes that were in the fourth instar. The consumption of B. thuringiensis by unparasitized larvae was shown to slow larval development.     

Performance of pine looper Bupalus piniarius larvae under population build-up conditions

In the first year of an outbreak, Bupalus piniarius larvae, encounter intense crowding. In the later stages of larval development, they are forced to feed on the non-preferred current-year needles of Scots pine or even on alternative hosts. It was hypothesized that larval feeding on a non-preferred resource (current-year needles) will negatively affect B. piniarius performance. It was also hypothesized that larval mutual interference (crowding without competition for food) will have negative additive effects. These hypotheses were tested in laboratory and field experiments. Fourth instar larvae were reared singly and under crowded conditions in cohorts of ten. Larvae in both situations were reared on control branches (containing both mature and current-year shoots) and branches containing only current-year shoots. Crowded larvae were reared also on Norway spruce, an alternative host. Crowding and feeding on a non-preferred resource had opposite effects on B. piniarius larval performance. Crowding in the late larval instars enhanced larval performance while absence of the preferred resource had negative effects. Larval growth rate was higher and development time was shorter for larvae exposed to crowded conditions than for solitary larvae. There was, however, no difference between the groups in final pupal weights or survival. Survival was 25% lower for larvae feeding on non-preferred current-year needles and pupal weights 9% lower, compared with results for larvae feeding on mature needles. Larvae feeding on Norway spruce suffered greatly extended development time, 82% lower survival, and resulted in 60% lighter pupae compared with conspecifics on Scots pine. It was concluded that not only quantity but also quality of the available food resource is critical for B. piniarius development.     

Larval mortality and development of Pseudoplusia includens (Lepidoptera: Noctuidae) reared on a transgenic Bacillus thuringiensis-cotton cultivar expressing CryIAc insecticidal protein.

The effects of a transgenic Bacillus thuringiensis (Bt)-cotton cultivar (DPL 32) on three instars of the soybean looper, Pseudoplusia includens (Walker), were determined in laboratory studies. First, third, and fifth instars were fed field collected Bt-cotton leaves for 1, 2, four and 7 d or until pupation, and then transferred to artificial diet. Mortality during the larval stage increased linearly in response to an increase in the length of feeding time on Bt-cotton by first and third instars. The maximum mortality of about two out of three larvae occurred for first instars fed on Bt-cotton until pupation. For the fifth instar, there was no significant response to feeding time; however, most of these larvae reached pupation before 4 d of feeding on Bt-cotton. The length of the larval developmental period also increased linearly with an increase in feeding time on Bt-cotton in first and third instars; again, there was no significant response in the fifth instars. For both mortality and larval developmental time, the linear trend lines for the first and third instars were quite similar. Pupal weight declined linearly in the first and fifth instars in response to feeding time on Bt-cotton. Although pupal weight also declined for third instars, the response was not linear. The effect of Bt-cotton appears not to extend past pupation in that there were no significant responses in mortality and developmental time of pupae during the pupal stage. These data indicate that larvae surviving Bt-cotton are adversely affected in several ways, which should be considered in evaluating Bt-cotton suppression of soybean looper infestations.     

Effect of dietary selenium supplementation on resistance to baculovirus infection

We have examined the effects of dietary selenium (Se) supplementation on larval growth and immunocompetence of the lepidopteran pest, the cabbage looper, Trichoplusia ni. Supplementation of the diet of T. ni larvae with 10–20 ppm Se resulted in a 1 day delay in pupation. The effects of the addition and/or removal of dietary Se on total Se bioaccumulation and sequestration were determined by neutron activation analysis of pupae. Early penultimate instar larvae moved from selenium containing diet to basal diet lost total pupal Se content down to the level of those fed basal diet. Conversely, larvae moved from basal diet to diet containing additional Se rapidly attained pupal Se levels comparable to larvae fed Se throughout larval development. Therefore, dietary Se is rapidly accumulated or lost during larval development, but significant amounts are sequestered from diet into pupae. Larvae were reared on diet supplemented with 5 or 10 ppm Se until the onset of the penultimate instar then infected per os with increasing concentrations of the fatal baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Larvae fed Se in the penultimate and ultimate instars were more resistant to viral infection than larvae not fed Se in the final instars. This study indicates that dietary Se levels rapidly impact Se assimilation and sequestration and that tissue Se levels are an important factor in resistance to AcMNPV infection in larval T. ni.     

Development of cost-effective medium for the large-scale production of a mosquito pupicidal metabolite from Pseudomonas fluorescens Migula

Although Bacillus thuringiensis and Bacillus sphaericus are being used extensively for mosquito control, the recent reports of development of resistance in insects against them prompted many workers to search for new microbial agents or their metabolites for mosquito control. This has resulted in the isolation of a new bacterial isolate Pseudomonas fluorescens Migula which is known to be toxic against larval and pupal stages of mosquitos and could be considered for further development as a biocontrol agent. But the large-scale production of this bacterium is expensive because the high cost of the medium restricts this bacterium is used in mosquito control programs. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw material Soya bean (Glycine max) by using 100-L bioreactor. Biomass and pupicidal metabolite production were satisfactory after P. fluorescens was produced on both media. Results showed that the Biomass in dry weight of Soya medium was 12.7 g/L and GPS medium (conventional medium) was 11.23 g/L. The maximum pupicidal metabolite production in Soya medium was (LC50 0.24 μL/mL) and in GPS medium was (LC50 0.44 μL/mL).     

RNA interference of an antimicrobial peptide, gloverin, of the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus thuringiensis

Gloverin is known to be an inducible antimicrobial peptide. This study reports a gloverin gene (Seglv) identified from the beet armyworm, Spodoptera exigua. Seglv encodes 175 amino acids with a signal peptide. Its amino acid sequence is highly homologous (>95%) to other known gloverins. Seglv was expressed from egg to adult stages even without immune challenge. Especially, in larval stage, it was expressed in all tested tissues, such as hemocyte, fat body, gut, and epidermis. However, the constitutive expression level was significantly elevated in response to bacterial challenge. Expression of a Toll gene was required for expression of Seglv. A recombinant Seglv protein was synthesized using a bacterial expression system and purified with an affinity chromatography. The recombinant protein showed a specific antibacterial activity against a Gram-positive bacterium, but no activity against a Gram-negative Escherichia coli. Injection of specific double stranded RNA (dsRNA) against Seglv could suppress its expression. Knockdown of Seglv expression induced a significant developmental retardation and resulted in hypotrophy pupae. The larvae treated with dsRNA were much more susceptible to Bacillus thuringiensis than the control larvae. These results suggest that Seglv acts as an antimicrobial peptide especially against Gram-positive bacteria including B. thuringiensis.     

Ecdysteroid-dependent protein synthesis in caste-specific development of the larval honey bee ovary

In the honey bee, Apis mellifera, the fifth larval instar is a critical period for caste differentiation. During this premetamorphic phase the hormonal milieu shows pronounced caste differences and several organs, particularly the ovaries, enter different developmental pathways leading to highly fertile queens and nearly sterile workers. Developmental profiles of total protein synthesis in larval ovaries showed marked caste differences starting with the early fifth instar. By two-dimensional electrophoresis, caste-specific patterns could be detected in the synthesis of a 29 kDa/pI 4.6 and two 24 kDa/pI 5.2–5.5. proteins (pI=isoelectric point). A marked decrease in the expression of these proteins was found to coincide with caste-specific differences in the haemolymph ecdysteroid titer. In vitro exposure of larval worker ovaries to physiological (10^–7 m) concentrations of synthetic makisterone A elicited an identical response. Juvenile hormone did not affect protein synthesis patterns in larval ovaries, and also did not inhibit or reverse the ecdysteroid-induced effects. Heat shock experiments revealed that the 29 kDa/pI 4.6 ecdysteroid-regulated protein belongs to the class of small heat shock proteins.     

Use of the egg parasiteEdovum puttleri [Hym.: Eulophidae] in an IPM system developed for Colorado potato beetle [Col.: Chrysomelidae] control on potatoes,Beltsville, Maryland

Edovum puttleri Grissell in conjunction with the fungal pathogenBeauveria bassiana (Balsamo) Vuillimen, a β-exotoxin ofBacillus thuringiensis varthuringiensis Berliner, and the insect growth regulator triflumuron (Alsystin 4F) were evaluated in a pest management system to control Colorado potato beetle,Leptinotarsa decemlineata (Say). Soil treatments ofB. bassiana had no significant impact on emerging Colorado potato beetle adults. Foliar applications of the β-exotoxin and triflumuron significantly reduced 1 ^st larval generation. Releases ofE. puttleri resulted in 56 % egg mass parasitization, significant reduction of 2 ^nd generation larvae, and significantly higher potato yields.Leptinotarsa texana (Schaeffer) was successfully used as an alternate host for production ofE. puttleri.      

Effect of Bacillus thuringiensis naturally colonising Brassica campestris var. chinensis leaves on neonate larvae of Pieris brassicae

The feeding of neonate larvae of Pieris brassicae (Order Lepidoptera) on leaves of brassica plants that had been colonised by Bacillus thuringiensis resulted in the death of 35% of the population within 72 h. The bacteria multiplied in the cadavers, resulting in an increase of about 50-fold compared to the living insects. Surviving insects showed no ill effects during the time of the study. There was negligible multiplication of B. thuringiensis in the frass.     

Development of a cost-effective medium for the large scale production of Bacillus thuringiensis var israelensis

Bacillus thuringiensis var israelensis (B.t.i.) has been widely used in mosquito control programs, but the large scale production of this bacillus is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max), groundnut cake powder (Arachis hypogea), and wheat bran extract (Triticum aestivum) by using 100-L fermentor. Sporulation, toxicity, and biomass were satisfactory after B.t.i. was produced on all the three media. Use of the soybean culture medium resulted in maximum toxicity (LC₅₀ 8.89 ng/ml against Culex quinquefasciatus IIIrd instar larvae), highest spore count (0.48 × 10¹¹ c.f.u./ml), and maximum biomass (7.8 g/L) within a short fermentation time of 24 h. Hence, this soybean-based culture medium was considered most economical for the large scale industrial production of B.t.i.     

Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae)

Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya chinensis (Thunberg), having a small number (2n♂=23) of large-sized chromosomes in males, was used for this purpose. The fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome aberrations induced by B.t. in the grasshoppers’ testicular cells showed that B.t. may induce chromosome aberrations, mainly chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose–effect relationships and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t. toxicity.     

Growth performance of Epirrita autumnata (Lepidoptera: Geometridae) on mountain birch: trees,broods, and tree x brood interactions

Summary The outcome of herbivore-host plant interactions is partly a function of variation within the two populations. We partitioned variance in herbivore growth performance into components attributable to differences between trees, differences between (full-sib) insect broods, and tree x brood interactions. Growth performance of Epirrita larvae feeding on a small (0.25 ha) population of mountain birch was greatly influenced by differences between individual trees. Up to 49% of the variation in insect growth rate was due to tree effects; 5th instar growth rates ranged from 0.38 to 0.56 mg·mg^-1·day^-1 across a sample of 8 trees. About 25% of the variation in pupal weights and larval periods was due to tree effects; on low quality trees larvae required a longer time to attain lower pupal weights. Differences between trees were also evident in physical and chemical characteristics of the leaves. Insect broods differed in the duration of the larval period (15% of the variance) which led to differences in the pupal weight attained (13% of the variance). However, brood-specific differences in growth rate were modest (6% in the 4th instar) or nonexistent (5th instar). There was no evidence for tree x brood interactions, which refutes the possibility of fine scale adaptation to particular tree phenotypes. Hypotheses to explain the existence of this variability, and to predict its evolutionary and ecological consequences, are advanced.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.