Extensive cultivation of crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has suppressed some major pests, reduced insecticide sprays, enhanced pest control by natural enemies, and increased grower profits. However, these benefits are being eroded by evolution of resistance in pests. We report a strategy for combating resistance by crossing transgenic Bt plants with conventional non-Bt plants and then crossing the resulting first-generation (F 1 ) hybrid progeny and sowing the second-generation (F 2 ) seeds. This strategy yields a random mixture within fields of three-quarters of plants that produce Bt toxin and one-quarter that does not. We hypothesized that the non-Bt plants in this mixture promote survival of susceptible insects, thereby delaying evolution of resistance. To test this hypothesis, we compared predictions from computer modeling with data monitoring pink bollworm (Pectinophora gossypiella) resistance to Bt toxin Cry1Ac produced by transgenic cotton in an 11-y study at 17 field sites in six provinces of China. The frequency of resistant individuals in the field increased before this strategy was widely deployed and then declined after its widespread adoption boosted the percentage of non-Bt cotton plants in the region. The correspondence between the predicted and observed outcomes implies that this strategy countered evolution of resistance. Despite the increased percentage of non-Bt cotton, suppression of pink bollworm was sustained. Unlike other resistance management tactics that require regulatory intervention, growers adopted this strategy voluntarily, apparently because of advantages that may include better performance as well as lower costs for seeds and insecticides.sustainability | evolution | resistance management | genetically modified | refuge G enetically engineered crops that produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been planted globally on a cumulative total of over 732 million ha since 1996 (1). The benefits of these Bt crops include pest suppression, reduced insecticide use, enhanced biological control, and increased farmer profits (2-7). However, increasingly rapid evolution of resistance to Bt crops by pests has eroded these benefits (8-11). The main strategy for delaying pest resistance to Bt crops aims to increase the survival of susceptible insects with "refuges" of host plants that do not produce Bt toxins (9). Although refuges can delay insect adaptation to Bt crops (2, 3, 9, 12), the optimal spatial scale for planting refuges remains unresolved (13-15). Also, because refuges are often perceived to cause short-term economic sacrifices for growers, they are usually imposed by regulations.Before 2010, regulations in the United States mandated refuges of non-Bt plants in blocks consisting of separate fields, rows, or strips (14). In 2010, the regulations were modified to include mixtures of Bt and non-Bt seeds generating a random array of Bt and non-Bt plants side by side within...

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying pest adaptation to Bt crops uses non-Bt host plants as “refuges” to increase survival of susceptible pests. To delay evolution of pest resistance to transgenic cotton producing Bt toxin Cry1Ac, the United States and some other countries have required refuges of non-Bt cotton, while farmers in China have relied on “natural” refuges of non-Bt host plants other than cotton. The “natural” refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005–2007 to 56% in 2008–2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.

Objective: To establish a method for noninvasive fetal cell isolation from maternal blood and prenatal testing of monogenic diseases by a combination of direct sequencing and targeted NGS-based SNP haplotyping from single fetal cells. Method: Peripheral blood of pregnant women in two families (congenital deafness and ichthyosis) was collected. After density-based separation and immunostaining with multiple biomarkers, candidate fetal cells were identified by high-throughput imagine analysis and picked up by automation. Individual fetal cells were subjected to STR-genotyping to identify their origin. Pathogenic mutations were identified by direct Sanger sequencing, and a combination of targeted NGS and SNP haplotyping using a custom panel. All the results were compared with amniotic fluid DNA. Results: Fetal trophoblasts were successfully harvested from maternal blood. STRgenotyping confirmed the fetal origin. Direct sequencing of pathogenic genetic mutations in fetal cells showed consistent results with amniotic fluid samples. For congenital deafness family, NGS-based SNP haplotyping also correctly identified the fetal haplotype. This single cell haplotyping method can be used to diagnose various genetic diseases. Conclusion: We have established a method for noninvasive prenatal testing of monogenic diseases from circulating trophoblast cells. This cell-based NIPT can be further applied to the prenatal diagnosis of various monogenic diseases.

The potential and propensity in ßight of Pectinophora gossypiella (Saunders) in the laboratory were measured using a 32-channel, computer-monitored ßight-mill system. Females ßew signiÞcantly farther within a 72-h ßight period than did males. The mean accumulated ßight distance and ßight duration of 1-d-old female individuals in a tethered-ßight test were 41.25 Ϯ 7.76 km and 23.87 Ϯ 2.55 h, respectively, whereas for male individuals, the same parameters were 23.46 Ϯ 2.13 km and 14.12 Ϯ 1.12 h. The ßight ability of adults was signiÞcantly positively correlated with pupal weight. The ßight activity of unmated pink bollworm moths increased daily from the time of eclosion, reaching a peak at 3Ϸ5 d and reducing gradually. All the moths could ßy normally at 16Ϸ36ЊC; however, the optimum temperature for ßight ranged from 24 to 28ЊC, whereas the optimum relative humidity ranged from 75 to 90%.

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The insecticidal crystal protein (Cry) genes of Bacillus thuringiensis are a key gene resource for generating transgenic crops with pest resistance. However, many cry genes cannot be expressed or form crystals in mother cells. Here, we report a novel Cry protein gene, cry65Aa1, which exists in an operon that contains a downstream gene encoding a hypothetical protein ORF2. We demonstrated that ORF2 is required for Cry65Aa1 expression and crystallization by function as a C-terminal crystallization domain. The orf2 sequence is also required for Cry65Aa expression, because orf2 transcripts have a stabilizing effect on cry65Aa1 transcripts. Furthermore, we found that the crystallization of Cry65Aa1 required the Cry65Aa1 C-terminus in addition to ORF2 or a typical Cry protein C-terminal region. Finally, we showed that Cry65Aa1 has a selective cytotoxic effect on MDA-MB231 cancer cells. This report is the first description of a 130-kDa mass range Cry protein requiring two C-termini for crystallization. Our findings reveal a novel evolutionary strategy of Cry proteins and provide an explanation for the existence of Cry protein genes that cannot form crystals in B. thuringiensis. This study also provides a potential framework for isolating novel cry genes from “no crystal” B. thuringiensis strains.

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