No abstract

f Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Nextgeneration sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5= and 3= tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity.

Laboratory feeding studies were carried out to determine the effects of the Bacillus thuringiensis (Berliner) CrylAb toxin on developmental time and mortality of Chrysoperla cameo (Stephens) larvae. A bioassay technique was developed that allowed for incorporation of the Cry lAb toxin into a liquid diet that was then encapsulated within small paraffin spheres. Because only 2nd and 3rd instars can penetrate the surface of the paraffin spheres, 2 different methods were used to rear chrysopid larvae through the 1st instar. The 1st method used small foam cubes soaked in non-encapsulated, liquid diet (with or without Cry lAb). The 2nd method used Ephestia kuehniella (Hiibner) eggs as prey during the first instar (no Cry lAb exposure). After reaching the 2nd ins tar, all larvae received encapsulated, artificial diet with or without Cry lAb, respectively. When reared only on artificial diet containing CrylAb toxin, total immature mortality was significantly higher (57%) than in the respective untreated control (30%). Also, significantly more chrysopid larvae died (29%) that received Cry lAb later during their larval development compared with the respective control (17%). Although mortality was consistently higher, no or only small differences in developmental times were observed between Cry lAb-treated and untreated C. cameo larvae. C. cornea larvae required significantly more time to complete larval development when reared on artificial diet only than when reared first on E. kuehniella eggs followed by encapsulated artifical diet or on only E. kuehniella eggs, regardless of exposure to Cry lAb. These results demonstrate that Cry lAb is toxic to C. cameo at 100 J.tmgl ml of diet by using encapsulated artificial diet.

Laboratory feeding experiments were carried out to study prey-mediated effects of artificial diet containing Bacillus thuringiensis proteins on immature Chrysoperla carnea. Activated Cry1Ab toxin and the protoxins of Cry1Ab and Cry2A were mixed into standard meridic diet for Spodoptera littoralis (Boisduval) larvae at the following concentrations; for Cry1Ab toxin, 25, 50, 100 µg g −1 diet were used; for Cry1Ab protoxin, the concentration was doubled (50 µg g −1 diet, 100 µg g −1 diet and 200 µg g −1 diet) to give relative comparable levels of toxin concentration. Cry2A protoxin was incorporated into the meridic diet at one concentration only (100 µg g −1 diet). For the untreated control, the equivalent amount of double distilled water was added to the meridic diet. Individual C. carnea larvae were raised on S. littoralis larvae fed with one of the respective treated meridic diets described above. The objectives were to quantify and compare the resulting effects on mortality and development time of C. carnea with those observed in two previous studies investigating prey-mediated effects of transgenic Cry1Ab toxin-producing corn plants and the other studying effects of Cry1Ab toxin fed directly to C. carnea larvae. Mean total immature mortality for chrysopid larvae reared on B. thuringiensis-fed prey was always significantly higher than in the control (26%). Total immature mortality of C. carnea reared on Cry1Ab toxin 100 µg g −1 diet-fed prey was highest (78%) and declined with decreasing toxin concentration. Cry1Ab protoxin-exposed C. carnea larvae did not exhibit a dose response. Prey-mediated total mortality of Cry1Ab protoxin-exposed chrysopid larvae was intermediate (46-62%) to Cry1Ab toxin exposed (55-78%) and Cry2A protoxin (47%) exposed C. carnea. In agreement with the previous studies, total development time of C. carnea was not consistently, significantly affected by the Bt-treatments except at the highest Cry1Ab toxin concentration. However, both highest mortality and delayed development of immature C. carnea raised on Cry1Ab toxin 100 µg g −1 diet -fed prey may have been confounded with an increased intoxication of S. littoralis larvae that was observed at that concentration. At all other B. thuringiensis protein concentrations S. littoralis was not lethally affected. Comparative analysis of the results of this study with those of the two previous studies revealed that in addition to prey/herbivore by B. thuringiensis interactions, also prey/herbivore by plant interactions exist that contribute to the observed toxicity of B. thuringiensis -fed S. littoralis larvae for C. carnea. These findings demonstrate that tritrophic level studies are necessary to assess the long-term compatibility of insecticidal plants with important natural enemies.

Selection of resistance in Spodoptera exigua (Hübner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryICresistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 g/g), whereas the 50% lethal concentration was 4.30 g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K d , and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.