Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.     

Toxicity of Bacillus thuringiensis to laboratory populations of the olive fruit fly (Dacus oleae).

A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus oleae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.     

Studies on bacterial flora in the population of the fall webworm, Hyphantria cunea Drury. (Lep., Arctiidae)

Abstract:  In this study, the bacterial flora of Hyphantria cunea Drury. (Lep., Arctiidae) were investigated during three hazelnut seasons from 1998 to 2000. Four different bacteria were found in dead and living larvae. They were isolated and identified as Bacillus thuringiensis , Escherichia freundii , Micrococcus sp. and Streptococcus sp. Laboratory experiments carried out to determine the insecticidal activities of these isolates showed that E. freundii and Micrococcus sp. did not have any insecticidal effect on second – third instar larvae of H. cunea . However, B. thuringiensis and Streptococcus sp. had 56 and 38% effects, respectively. Crystals and spores from B. thuringiensis were also purified and the crystals, spores and crystals–spore mixture were tested separately against the larvae of H. cunea . It was found that the insecticidal activities of the crystals, spores and crystal–spore mixture were 37.5, 25 and 62.5%, respectively, on second – third instar larvae of H. cunea . These results indicate that the crystal–spore mixture has 6.5% more insecticidal effect than that of the vegetative cells of the B. thuringiensis isolate.     

Influence of Bacillus thuringiensis var. israelensis on oviposition of Aedes albopictus (Skuse).

The effect of Bacillus thuringiensis var israelensis (B.t.i.) on the oviposition behavior of Aedes albopictus was evaluated in the field and laboratory in Clemson, SC, U.S.A. In the field, water taken from containers in which mosquito larvae were reared (conditioned water) was placed in 16 containers. Eight containers received 50 jld of B.t.i., and eight with water only were kept as controls. In the laboratory, field-collected females of Ae. albopictus were placed in rearing chambers and provided two containers for oviposition, one with 50 microl of B.t.i and one a control with water only. Eight cage experiments were conducted, five using filtered tap water and three with conditioned water. In the field over the 13 trials, more eggs were laid in the containers with B.t.i. although no significant difference was found in the number of eggs between the treatment and controls over 72 h. In the laboratory, more eggs were laid in the containers with B.t.i. versus the controls. The containers had filtered tap water and B.t.i. had significantly more eggs laid in them compared to the controls.     

Insecticidal activity of strains of Bacillus thuringiensis on larvae and adults of Bactrocera oleae Gmelin (Dipt. Tephritidae).

The olive fly, Bactrocera oleae, is the key pest on olives in the Mediterranean area. The pest can destroy, in some cases, up to 70% of the olive production. Its control relies mainly on chemical treatments, sometimes applied by aircraft over vast areas, with their subsequent ecological and toxicological side effects. Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects, including the orders of Lepidoptera, Diptera, and Coleoptera and other invertebrates. The aim of this study was to search for isolates toxic to B. oleae. Several hundred B. thuringiensis isolates were obtained from olive groves and olive presses in different areas of Greece, Sardinia (Italy), and Spain and from cooperating scientists throughout the world. Some isolates were found toxic only to adults or larvae and some to both stages of the olive fly. In addition, the most toxic isolates were assayed on Opius concolor Szepl. (Hym. Braconidae), the most important parasitoid of the olive fruit fly. Only 3 isolates out of 14 gave significant mortality against this parasitoid. Several of the most toxic crystalliferous isolates may contain novel toxins since they gave no PCR products when probed with primers specified for 39 known toxin genes.     

桔小实蝇对石榴的产卵偏好性研究

明确石榴品种、成熟度以及果实的完整性对桔小实蝇产卵偏好的影响,可为石榴园桔小实蝇的行为调控提供重要理论依据。本研究以采自四川省会理市彰冠镇不同品种、不同成熟度石榴果实作为材料,通过选择性试验,统计果实上的产卵孔数和着卵量,分析桔小实蝇的产卵选择行为。研究结果显示,桔小实蝇对不同品种石榴的产卵偏好性为软籽石榴>硬籽石榴>野生硬籽石榴;对不同成熟度石榴的产卵偏好性为熟果>完熟果>生果;石榴裂果比正常果更吸引桔小实蝇产卵。相关性分析结果表明,在石榴正常果状态下,软籽石榴和野生硬籽石榴的可溶性固形物含量与产卵孔数、着卵量均呈显著正相关性,而硬籽石榴可溶性固形物含量与产卵孔数、着卵量均无显著相关性;在石榴裂果状态下,各石榴品种可溶性固形物含量和着卵量均无显著相关性。桔小实蝇更偏好于软籽石榴熟果、裂果以及可溶性固形物含量为14.00%～17.00%的果实上产卵,会理地区8-9月时石榴的生长处于熟果期且多裂果,因此该时期应为桔小实蝇发生为害的关键时期,要提前做好防控。     

香茅精油对桔小实蝇产卵驱避作用及其化学成份分析

研究了香茅Cymbopogon nardus精油对桔小实蝇Bactrocera dorsalis (Hendel)的产卵驱避作用及其化学成分。结果表明,香茅精油对桔小实蝇产卵具有一定的驱避作用,不同浓度香茅精油处理芒果上的产卵量均显著低于对照,且随着香茅精油浓度的提高其产卵量逐渐减少,当浓度为10000μg/mL时,产卵驱避率为70.06%。采用气相色谱—质谱联用(GC-MS)技术分析了香茅精油的化学成份,结果发现该精油挥发物中含量较高的是3-蒈烯,香茅醛,香叶醇,香茅醇,柠檬烯。     

Selection, egg viability, and fecundity of the sugarcane borer (Lepidoptera: Crambidae) with tebufenozide.

Two separate attempts to select the sugarcane borer, Diatraea saccharalis (F.), for resistance to tebufenozide were unsuccessful. Both selected colonies were lost after the fourth generation due to a lack of oviposition. Differences were not detected in fecundity or percent egg viability for 5-d-old third instars exposed to concentrations (EC5, EC15, and EC30) of tebufenozide for 7 d. Decreases (P < or = 0.01) in mean female pupal weights were detected in larvae exposed to EC15 and EC30 concentrations. An ovicidal impact using serial dilutions of tebufenozide (10, 100, and 200 ppm) also was detected. Percent viability was reduced from 98% for untreated eggs to 61% for eggs dipped in 10 ppm and below 6% for eggs dipped in > or = 100 ppm. Eggs treated with 200 ppm did not hatch. Though some embryonic development was observed on eggs treated with the high concentrations (100 and 200 ppm), sclerotization of head capsule was not apparent. The ovicidal property of tebufenozide may enhance its effectiveness in controlling populations of the D. saccharalis on an area-wide basis. Fecundity and egg viability were affected in later generations of selection; however, separate studies assessing individuals that were exposed to sublethal concentration (EC5, EC15, and EC30) of tebufenozide as third instars for 7 d in one generation did not detect differences.     

Pathogenicity of intrathoracically administrated Bacillus thuringiensis spores in Blatta orientalis

The ability of Bacillus thuringiensis to produce septicaemia in Periplaneta americana and Blatta orientalis has been investigated. Spores and crystals from several wild-type strains as well as spores of a B. thuringiensis crystal-deficient mutant, were first orally administrated at high doses, and no significant mortality was recorded. Intrathoracic injection of spore suspensions in P. americana revealed that this species is not very susceptible to B. thuringiensis spores. B. orientalis, by contrast, was found to be very susceptible to B. thuringiensis, with a LD(50) of about 35,000 spores, that is similar to that reported on Lepidoptera challenged with parenterally injected B. thuringiensis.     

Effects of forced egg-retention in Aedes albopictus on adult survival and reproduction following application of DEET as an oviposition deterrent.

The insect repellent DEET (0.1% concentration), used as a mosquito oviposition deterrent in the laboratory, influenced the retention and maintenance of mature eggs by caged gravid female Aedes albopictus Skuse. This egg-retention mechanism could benefit survival because the gravid females were ultimately able to lay maintained eggs upon availability of water, but the length of forced egg-retention time reduced the number of eggs laid per female. Gravid females with retained eggs also laid a higher percentage of eggs that failed to tan, and this percentage increased with time duration of egg-retention. Percent egg hatch was not significantly affected by DEET when used as an oviposition deterrent; however, percent hatch was affected by time duration of egg-retention in both treated (exposed to DEET) and untreated (control) gravid females. The rate of egg hatch was considerably reduced after three weeks of retention; this reduction declined to zero for treated and control females at six and four weeks post-treatment, respectively. The fecundity and fertility of gravid female Ae. albopictus were affected by the time duration of forced egg-retention.     

Gene clusters located on two large plasmids determine spore crystal association (SCA) in Bacillus thuringiensis subsp. finitimus strain YBT-020

Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named 'spore crystal association' (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.     

Density and structure of Saissetia oleae (Hemiptera: Coccidae) populations on citrus and olives: relative importance of the two annual generations

Saissetia oleae (Olivier) (Hemiptera: Coccidae) populations were studied and compared in citrus (Citrus spp.) and olive (Olea europaea L.) groves to determine the number of generations, crawler emergence periods and changes in population density during the year. Ten citrus and four olive groves were sampled regularly between March 2003 and December 2005 in eastern Spain, covering an area of 10,000 km2. Each sample consisted of 16 branches and 64 leaves. Saissetia oleae populations presented a similar trend in both crops during the three years of study. Populations peaked in July, when crawlers emerged after the egg-laying period, and decreased during several months due to mortality of first instars in summer. A second crawler emergence period, with lower numbers and more variability from year to year, occurred between October and March. Populations did not increase during this period, probably because most eggs and crawlers perished during the winter and also because females that gave rise to this fall-winter generation were half as big and fecund as spring females. No differences were found between the size of mature females that had developed on citrus and on olives during the spring. Considering this population pattern, the best seasonal period to apply pesticides to control S. oleae would be at the end of July, when populations are synchronous, all crawlers have already emerged, and first instars predominate.     

Isolation of Multiple Subspecies of Bacillus thuringiensis from a Population of the European Sunflower Moth, Homoeosoma nebulella

Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of H. nebulella showed that the isolates of B. thuringiensis subspp. aizawai, kurstaki, and thuringiensis were the most toxic, with 50% lethal concentrations (LC(inf50)s) of 0.15, 0.17, and 0.26 (mu)g/ml, respectively. The isolates of B. thuringiensis subspp. morrisoni and thompsoni had LC(inf50)s of 2.62 and 37.5 (mu)g/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of B. thuringiensis producing different insecticidal proteins.     

Stimuli affecting selection of oviposition sites by female peach twig borer, Anarsia lineatella Zeller (Lepidoptera: Gelechiidae)

In laboratory and field experiments, stimuli were tested that might affect oviposition decisions by female peach twig borer moths, Anarsia lineatella Zeller (Lepidoptera: Gelechiidae). When given a choice between immature green peach fruits, green mature peach fruits and soft-ripe peach fruits, the latter received the fewest eggs. Fuzzy halves of peach fruits received ten times more eggs then shaved hairless halves. Volatiles from both almond and peach shoots induced more oviposition by females than by control stimuli. Similarly, volatiles from immature green peach fruits, mature green or mature hard-ripe peach fruits induced more oviposition than their respective control stimuli. In a choice experiment, volatiles from immature peach fruit stimulated three times more oviposition than those from soft-ripe peach fruit. Discrimination against mature soft-ripe peach fruits as potential oviposition sites may lie in the phenology of A. lineatella and host peach fruits. Larval development to the pupal stage takes 15–27 days. Therefore, any eggs laid on a ripe fruit 14 days before it falls from the tree will not likely develop into adult insects because developing larvae will only reach third or fourth instar before the fruit is decomposed, and only first and second instar larvae can overwinter.     

Unique regulation of crystal protein production in Bacillus thuringiensis subsp. yunnanensis is mediated by the cry protein-encoding 103-megadalton plasmid.

In sporulating cultures of Bacillus thuringiensis subsp. yunnanensis HD977, two cell types are observed: cells forming only spores and cells forming only crystals. Curing analysis suggested that the crystal proteins are plasmid encoded. Through plasmid transfer experiments, it was established that a 103-MDa plasmid is involved in the crystal production. Conjugal transfer of this plasmid to Cry- recipient cells of Bacillus thuringiensis subsp. kurstaki HD73-26 conferred the ability to produce crystals exclusively on asporogenous cells of the recipient, indicating that the 103-MDa plasmid mediates the unique regulation of Cry protein production. When the dipteran-specific cryIVB gene was introduced into wild-type (Cry+) and Cry- backgrounds of B. thuringiensis subsp. yunnanensis by phage CP51ts45-mediated transduction, similar to all other B. thuringiensis strains, irregular crystals of CryIVB protein were produced by spore-forming cells in both backgrounds. However, the synthesis of the bipyramidal inclusions of B. thuringiensis subsp. yunnanensis was still limited only to asporogenous cells of the transductant. Thus, it appears that the unique property of exclusive crystal formation in asporogenous cells of B. thuringiensis subsp. yunnanensis is associated with the crystal protein gene(s) per se or its cis acting elements. As the crystals in B. thuringiensis subsp. yunnanensis were formed only in asporogenous cells, attempts were made to find out whether crystal formation had any inhibitory effect on sporulation. It was observed that both Cry+ and Cry- strains of B. thuringiensis subsp. yunnanensis (HD977 and HD977-1, respectively) exhibited comparable sporulation efficiencies. In addition, the Cry- B. thuringiensis subsp. kurstaki host (HD73-26) and its Cry+ transconjugant (HD73-26-16), expressing the B. thuringiensis subsp. yunnanensis crystal protein, were also comparable in their sporulation efficiencies, indicating that production of the crystal proteins of B. thuringiensis subsp. yunnanensis does not affect the process of sporulation.     

矿物油乳剂作用下橘小实蝇的产卵拒避及触角电位反应

为了研究矿物油乳剂对害虫行为的干扰作用,测定和比较了4种矿物油乳剂对橘小实蝇Bactrocera dorsalis (Hendel)的产卵拒避效果及在其作用下该虫的触角电位反应（EAG）,并测试了其中拒避效果最好的Caltex Lovis夏用油的不同施用方式（浸蘸法和喷雾法）及不同施用浓度（150,250,350,450倍液）对橘小实蝇产卵拒避的影响。结果显示：分别用4种矿物油乳剂200倍液以浸蘸法处理香蕉,仅Caltex Lovis夏用油处理对橘小实蝇产卵拒避效果显著,其平均产卵孔数仅为清水对照的15.14%;其他3种矿物油乳剂则没有明显效果。这表明矿物油乳剂对昆虫行为的干扰作用与其组成成分相关。4种矿物油乳剂对橘小实蝇的产卵拒避效果与各矿物油乳剂的50倍液滴于滤纸条后0.5 h的雌成虫EAG值间无显著相关性,但与滴于滤纸条后3 h（滤纸条上水分已蒸发）的EAG值间有显著的负相关性,说明矿物油乳剂的挥发性组分对橘小实蝇的产卵拒避影响不大,因此推测矿物油乳剂在植物表面形成的油膜对植物挥发性物质的封闭作用与其产卵拒避效果可能有一定的关系。浸蘸法不同浓度处理间的产卵拒避效果无显著差异,均明显高于喷雾法处理及清水对照。在喷雾法处理中,仅150倍液有显著产卵拒避效果。这说明,矿物油乳剂需要在植物表面达到一定的沉积量,才能对橘小实蝇有较好的产卵拒避作用。与喷雾法相比,浸蘸法可能较利于矿物油在植物表面的沉积,这也佐证了矿物油乳剂所形成油膜的封闭性与其产卵拒避效果有一定的关系。     

Embryonic Development and Rates of Metabolic Activity in Early and Late Hatching Eggs of the Major Malaria Vector Anopheles gambiae

Anopheles gambiae eggs generally hatch at the completion of embryo development; two-three days post oviposition. However, staggered or delayed hatching has been observed whereby a single batch of eggs shows marked variation in time-to-hatch, with some eggs hatching 18 days post oviposition or later. The mechanism enabling delayed hatch has not been clearly elucidated but is likely mediated by environmental and genetic factors that either induce diapause or slow embryo development. This study aimed to compare metabolic activity and embryonic development between eggs collected from sub-colonies of the baseline Anopheles gambiae GAH colony previously selected for early or late time-to-hatch. Egg batches from early and late hatch sub-colonies as well as from the baseline colony were monitored for hatching. For both time-to-hatch selected sub-colonies and the baseline colony the majority of eggs hatched on day two post oviposition. Nevertheless, eggs produced by the late hatch sub-colony showed a significantly longer mean time to hatch than those produced by the early hatch sub-colony. The overall proportions that hatched were similar for all egg batches. CO₂ output between eggs from early and late hatch sub-colonies showed significant differences only at 3 and 7 days post oviposition where eggs from the early hatch and the late hatch sub-colony were more metabolically active, respectively. No qualitative differences were observed in embryo development between the sub-colonies. It is concluded that all viable embryos develop to maturity at the same rate and that a small proportion then enter a state of diapause enabling them to hatch later. As it has previously been shown that it is possible to at least partially select for late hatch, this characteristic is likely to involve genetic as well as environmental factors. Delayed hatching in An. gambiae is likely an adaptation to maximise reproductive output despite the increased risk of desiccation in an unstable aquatic environment.     

Oviposition attractancy of bacterial culture filtrates: response of Culex quinquefasciatus

Oviposition attractants could be used for monitoring as well as controlling mosquitoes by attracting them to lay eggs at chosen sites. In the present study, culture filtrates of seven bacterial species were tested for their attractancy against gravid females of Culex quinquefasciatus. When their oviposition active indices (OAI) were studied, the culture filtrates of Bacillus cereus and Pseudomonas fluorescens exhibited oviposition attractancy (OAI = > 0.3) at 100 ppm and the OAI were respectively 0.70 and 0.47. Culture filtrates of B. thuringiensis var. israelensis (wild type), B. t. var. israelensis (mutant) and B. sphaericus showed attractancy at 2000 ppm with OAI of respectively 0.71, 0.59 and 0.68. However, the OAI of B. megaterium as well as Azospirillum brasilense was 0.13 (at 2000 ppm), which was less than 0.3 required to be considered them as attractants. When the oviposition attractancy of the bacterial culture filtrates were compared with that of a known oviposition attractant, p-cresol (at 10 ppm), the culture filtrates of B. t. var. israelensis (wild type) and B. cereus were found to be more active than p-cresol, respectively with 64.2 and 54.3% oviposition.