Inheritance of cold shock tolerance in hybrids of Drosophila virilis and Drosophila lummei

The genetic basis of the difference in cold shock tolerance between the southern temperate Drosophila virilis and its boreal relative D. lummei is studied. After adult eclosion, the parental stocks, reciprocal F₁ and backcross hybrids were pretreated for eight days at 18°C or at 6°C. The cold shock used consisted of fast cooling to-10°C and exposure to this temperature for varying lengths of time. D. lummei tolerated such exposure for 40–50% longer than did D. virilis (100–135% after acclimation). Reciprocal F₁ females, differing only in their maternal cytoplasm deviated significantly from each other, and the reciprocal F₁ males even more so, the contribution of the X chromosome being three to four times that of the cytoplasm. The cold resistance scores of the hybrid males were more extreme than those of the parental stocks. Autosomally heterozygous males with the X chromosome and cytoplasm of virilis were the weakest flies studied. The reciprocal males (X chromosome and cytoplasm of lummei) survived better than the parental lummei stock. The reciprocal differences decreased after cold temperature acclimation. The roles of the four major autosomes were analyzed by backcrossing the reciprocal F₁ males with females of the virilis marker stock. The third chromosome of lummei as heterozygous contributed most to cold tolerance, while the other autosomes had a rather weak effect in the opposite direction (virilis homozygotes survived better), which disappeared after acclimation at 6°C. Some of the cold susceptibility of F₁ hybrids disappeared in chromosomally identical backcross flies, indicating complex cytoplasmchromosomal interactions.     

Sperm competition in the Drosophila female

Summary Modified B ^S translocation males were developed at 26.0° C where univalentbearing gametes are recovered with less than half the frequency than at 18.0° C. Upon eclosion the males were stored for definite time periods at either temperature before mating individually to single y free-X females. the transfer cultures of the females show a higher frequency of recovery of univalent-bearing progeny regardless of the temperature or storage treatment of the male. In addition, postmeiotic temperature treatment does not appear to fundamentally alter the overall frequency of recovery of univalent-bearing gametes which is presumably determined by the developmental temperature of the male. A similar trend is observed for matings of y females to single X.Y^SY^L/O males in which the males were developed and stored at 26.0° C; namely, a higher frequency of recovery of attached-XY gametes in the transfer cultures.     

On the Adaptive Nature of the Dissociation Process in Bacillus thuringiensis

The process of dissociation into variants that differ in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40°C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

Recoverability of heat-injured Bacillus spores by lysozyme and EDTA or alkaline thioglycollate

The D_95°C value of Bacillus thuringiensis spores plated in the presence of lysozyme increased from 3.0 min to 3.6 min by post-treatment of heat-injured spores with 50mm EDTA. In the case of Bacillus alvei and Bacillus polymyxa spores D-values decreased from 4.9 to 4.3 min and from 4.7 to 4.1 min respectively. Post-treatment of heat-injured spores treated with alkaline thioglycollate increased D_95°C values of Bacillus alvei from 4.2 to 5.3 min, B. thuringiensis 3.6 to 4.7 min, and Bacillus polymyxa from 4.2 to 5.0 min when spores were plated in the presence of lysozyme. Electron micrographs of heat-injured B. alvei spores treated with sodium thioglycollate indicated that the coat layers of the treated spores were granulated and less intact than the control spores.     

Altered mating behaviour in a Cry1Ac-resistant strain of Helicoverpa armigera (Lepidoptera: Noctuidae)

Randomness of mating between susceptible and resistant individuals is a major factor that closely relates to the refuge strategy of resistance management for Helicoverpa armigera (Hübner) to Bacillus thuringiensis cotton. The mating behaviour of Cry1Ac‐susceptible and Cry1Ac‐resistant strains of H. armigera was compared to investigate the randomness of their mating. The percentage of mating was lower for Cry1Ac‐resistant H. armigera compared with that of the susceptible strain under both no‐choice and multiple‐choice conditions. The low percentage of mating in the resistant strain indicates a reduced incidence of successful mating. The percentage of spermatophore‐containing mated female H. armigera in the crossing of susceptible females × resistant males was significantly lower than in the crossing of resistant females × susceptible males, but the observed mating frequencies of these two types of cross were similar to each other. This indicates that resistant males reduce the incidence of mating paternity more than they do their mating frequency. The percentages of heterogametic matings (susceptible females × resistant males, resistant females × susceptible males) in the multiple‐choice experiment were lower than those of homogametic matings (susceptible × susceptible, resistant × resistant) on peak mating nights. However, the difference between heterogametic and homogametic mating was not significant, indicating that there was a random mating between susceptible and resistant strains. The results presented here do not reflect reality in mating associated with Cry1Ac resistance but can provide insight into variable expression.     

Action of Cu2+ on Bacillus thuringiensis Growth Investigated by Microcalorimetry

By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism is determined at 28°C and the effect of Cu²⁺ on B. thuringiensis growth is studied. Copper is regarded as an essential trace element for life. Its deficiency may be the cause of diseases. Cu²⁺ at different concentrations has different effects on B. thuringiensis growth metabolism: a low concentration (0–30 g/ml) of Cu²⁺ can promote the growth of B. thuringiensis, a high concentration (40–120 g/ml) can inhibit growth of the bacteria, and a concentration of Cu²⁺ of up to 130 g/ml completely inhibits B. thuringiensis growth.     

Thermal acclimation in the grain miteAcarus siro (Acari: Acaridae)

Grain mites reared on wheat germ at 21±1°C and 85% r.h. were treated (acclimated) as adults at 0, 14, 28 or 33°C for 1 or 4 days. Fecundity, longevity and numbers of F₁ females and males produced were compared. A treatment of 1 day was enough to acclimateAcarus siro and change the studied developmental parameters. Acclimation always lowered fecundity but the magnitude of the effect was dependent upon the temperature regime. The number of males and females emerging differed greatly among various temperature regimes; however, the sex ratio of emerged adults remained in most instances quite constant.     

UBER DEN BIOTEST VON BACILLUS THURINGIENSIS-EXOTOXIN MIT DROSOPHILA MELANOGASTER

Summary Larvae of Drosophila melanogaster (Meigen), placed as eggs on the substrate, are suitable for bioassay of the toxic qualities of the supernatant from culture media of Bacillus thuringiensis Berliner. Only supernatants of active strains of B. thuringiensis var. thuringiensis contained a thermostable exotoxin and, therefore, caused significant mortality. Supernatants prepared from cultures of other varieties of B. thuringiensis, however, as well as from cultures of Bacillus cereus had no effect (Tab. I). Pure preparations of the spore-endotoxin-complex of B. thuringiensis ded not kill larvae of Drosophila melanogaster even in doses of 0.16 per cent of the medium (Tab. II). According to the qualitative classification of commercial B. thuringiensis-preparations by Burgerjon, Biospor 2802 (Farbwerke Hoechst, Germany), belongs to group I, whereas Bakthane L-69 (Rohm & Haas, USA) is representative of group II.

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Über den Biotest von Bacillus thuringiensis-Exotoxin mit drosophila melanogaster

Zusammenfassung Zur Prüfung der toxischen Qualitäten von Kulturüberständen von Bacillus thuringiensis-Kulturen dienten Larven von Drosophila melanogaster. Es wurden abgezählte mengen von Eiern auf das kontaminierte Substrat aufgebracht und am Ende des Versuchs die entwickelten Imagines registriert. Nur Überstände aktiver Stämme von B. thuringiensis var. thuringingiensis enthielten ein thermostabiles Exotoxin und bewirkten eine entsprechende Mortalität. Kultur-überstände anderer Varietäten von B. thuringiensis wie auch von Bacillus cereus hatten keine Wirkung (Tab. I). Gereinigte Präparate des Sporen-Endotoxin-Komplexes von B. thuringiensis waren selbst in Dosen von 0,16% des Mediums unwirksam (Tab. II). Entsprechend der qualitativen Klassifikation von industriellen B. thuringiensis-Präparaten nach Burgerjon gehört Biospor 2802 (Farbwerke Hoechst, Deutschland) zur Gruppe I, während Bakthane L-69 (Rohm & Haas, USA) für die Gruppe II repräsentativ ist.

     

Isolation and characterization of Bacillus thuringiensis strains from olive-related habitats in Turkey

Aims: To isolate Bacillus thuringiensis strains from different olive‐related habitats (olive groves and olive oil factories) in Turkey and to characterize these strains by molecular methods. Methods and Results: A total of 150 samples, consisting of olive grove soil, green olive leaves, olive leaf residues, animal faeces, olive pomace and dust, were examined for the presence of B. thuringiensis. One hundred B. thuringiensis strains were isolated from 54 environmental samples (36%) and characterized in terms of crystal morphology, cry and cyt gene content by polymerase chain reaction, plasmid profiles and 16S‐internal transcribed spacer ribosomal DNA restriction fragment length polymorphism (16S‐ITS rDNA RFLP). The highest percentage of samples containing B. thuringiensis was found in 38 out of 54 total soil samples (70%). Of the 100 B. thuringiensis isolates, the most frequent crystal shapes were irregularly shaped (24%), spherical‐irregular pointed (19%), cuboidal (17%) and spherical (16%). The cry1 plus cry4 genotype was the most abundant genotype in our collection (21%). RFLP analysis of the amplified 16S‐ITS rDNA revealed 11 distinct patterns for the isolates and 10 reference strains. Conclusions: Bacillus thuringiensis isolates showed a great genetic diversity and crystal shape heterogeneity. Significance and Impact of the Study: This is the first study on the isolation and characterization of B. thuringiensis from olive‐related habitats in Turkey. No correlation was observed between the cry genotypes and insecticidal crystal shapes of the isolates. Restriction profiles of 23% of the isolates were found to be different from those of the 10 reference strains used.     

Genetic variation in fitness parameters associated with resistance to Bacillus thuringiensis in male and female Trichoplusia ni

Resistance of insects to insecticides is often associated with reduced fitness in the absence of selection. We examined fitness trade-offs associated with resistance to the microbial insecticide, Bacillus thuringiensis (Bt), across full-sib families in a resistant population of Trichoplusia ni. Significant genetic variation in and heritability of susceptibility to Bt occurred among the full-sib families. Male pupal weight was positively correlated with Bt susceptibility, indicating a potential fitness cost, but no such correlation occurred for females. Significant heritability for pupal weight was present for males but not females. A significant negative genetic correlation existed between development time and Bt susceptibility, indicating that resistant larvae developed more slowly than susceptible larvae. Selection for Bt resistance in T. ni resulted in changes in life-history traits that affected males more than females.     

Construction of an <Emphasis Type="Italic">Escherichia coli</Emphasis> to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> shuttle vector for large DNA fragments

Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research.     

Effects of caffeine on chromosomal loss and nondisjunction in Drosophila melanogaster

Three types of male larvae, normal X males and two types with structurally abnormal X chromosomes (ring X and short X sc ⁴ sc ³, y) were treated during the third instar with 0.5 per cent caffeine in nutrient medium. Upon eclosion, these males were mated to yellow and Oregon-R wild type females. The F₁ generation of each cross was scored for normal (XX and XY) and abnormal (XO and XXY) progeny. Statistical analyses of data demonstrate that caffeine increases chromosomal loss for all genotypes tested. The effect of caffeine on nondisjunction, however, is not clear. There are at least marginal increases in all cases when Oregon-R females are used. Slight increase and decreases noted for offspring of yellow females appear to be dependent upon the genotype of the inseminating male.     

Biological characterization of two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains toxic against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT₅₀. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.     

Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.     

The apple sawfly, Hoplocampa testudinea: a temperature driven model for spring emergence of adults

The effect of temperature on post-diapause development of the apple sawfly Hoplocampa testudinea Klug was studied under controlled conditions. Survival was found to decrease at temperatures higher than 15°C, the lethal temperature being close to 25°C. The lower thermal threshold was 4.5°C; the values for males and females did not differ significantly. However, different values were established for unprotected individuals, in vermiculite and in potting compost respectively. Based on the overall mean and variance of the developmental time and the overall thermal threshold a simple soil temperature driven phenology model was constructed using a time- varying distributed delay. The validation with independent field and semi-field data revealed the model to be a reliable tool to predict apple sawfly phenology and hence to determine the optimal time for the installation of white sticky traps or for control measures such as insecticide treatments.     

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.Non-standard abbreviations BSA bovine serum albumin - CAT chloramphenicol acetyltransferase - Cm^R chloramphenicol resistant - PAB Penassay broth - SDS sodiumdodecylsulfate - Tc^R tetracycline resistant     

Developmental genetics of a temperature-sensitive RNA polymerase II mutation in Drosophila melanogaster

Summary Purified RNA polymerase II (RNA nucleotidyl-transferase; EC 2.7.7.6) extracted from flies possessing lesions in the Ultrabithorax-like (Ubl) locus of Drosophila melanogaster has altered activity in vitro (Greenleaf et al. 1979, 1980; Coulter and Greenleaf 1982). This strongly suggests that the Ubl locus encodes a subunit of RNA polymerase II. Ethyl methanesulfonate was used to induce a temperature-sensitive mutation in this locus. Flies either homozygous or hemizygous for this new X–linked mutation (Ubl ^ts) display viability comparable to that of wild-type flies at 22° C but are lethal at 29° C. The temperature-sensitive period for Ubl ^ts flies is between gastrulation (6 h, 29° C) and pupation (9–10 days, 22° C). Zygotes shifted from 22° C to 29° C die at either the late embryonic or first larval instar stage while temperature shifts of second and third instar larvae result in the lethal phase occurring at the pupal stage. Most pupae shifted from 22° C to 29° C undergo metamorphosis and eclose as adults. Adults are viable if placed at 29° C; however, all females and some males become sterile if maintained at this temperature.Somatic recombination was used to induce clones homozygous for a null allele of Ubl at different stages of development. Clones of this null allele appear to be cell lethal indicating that the Ubl ⁺ gene product is required at all stages of development. The viability of Ubl ^ts pupae and adults at 29° C may result from only a partial reduction in activity caused by the mutation at this nonpermissive temperature.     

Survival of a strain of Bacillus megaterium carrying a lepidopteran-specific gene of Bacillus thuringiensis in the phyllospheres of various economically important plants

The colonizing ability of a transcipient strain of Bacillus megaterium carrying a lepidopteran-specific cryIA (a) gene of Bacillus thuringiensis in the phyllospheres of various economically important plants was studied. Similar experiments were also carried out using the parental B. thuringiensis var. kurstaki strain HD1 for a comparison. While the transcipient remained on the leaves of cotton and okra for more than 28 days, its survival in phyllospheres of mulberry, peanut, chickpea, tomato and rice was rather limited to about 3 – 5 days. The persistence of B. thuringiensis, on the other hand, was extremely short (i.e. less than 4 days) on all the crop plants tested.     

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).