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Background: Transgenic insect-resistant rice offers an environmentally friendly approach to mitigate yield losses caused by lepidopteran pests, such as stem borers. Bt (Bacillus thuringiensis) genes encode insecticidal proteins and are widely used to confer insect resistance to genetically modified crops. This study investigated the integration, inheritance, and expression characteristics of codon-optimised synthetic Bt genes, cry1C* and cry2A*, in transgenic early japonica rice lines., Methods: The early japonica rice cultivar, Songgeng 9 (Oryza sativa), was transformed with cry1C* or cry2A*, which are driven by the ubi promoter via Agrobacterium tumefaciens-mediated transformation. Molecular analyses, including quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Southern blot analysis were performed to confirm transgene integration, inheritance, transcriptional levels, and protein expression patterns across different tissues and developmental stages., Results: Stable transgenic early japonica lines exhibiting single-copy transgene integration were established. Transcriptional analysis revealed variations in Bt gene expression among lines, tissues, and growth stages, with higher expression levels observed in leaves than in other organs. Notably, cry2A* exhibited consistently higher mRNA and protein levels than cry1C* across all examined tissues and developmental time points. Bt protein accumulation followed the trend of leaves > stem sheaths > young panicles > brown rice, with peak expression during the filling stage in the vegetative tissues., Conclusions: Synthetic cry2A* displayed markedly elevated transcription and translation compared to cry1C* in the transgenic early japonica rice lines examined. Distinct spatiotemporal patterns of Bt gene expression were elucidated, providing insights into the potential insect resistance conferred by these genes in rice. These findings will contribute to the development of insect-resistant japonica rice varieties and facilitate the rational deployment of Bt crops., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The ladybeetle Propylea japonica is a widely distributed natural enemy in many agricultural systems. P. japonica is often used as a test organism for safety assessments of transgenic Bacillus thuringiensis crops. Plant varieties expressing the Vip3Aa insecticidal protein are not currently commercially available in China. In this study, protease inhibitor E-64 was used as a positive control to examine the responses of P. japonica larvae to a high concentration of Vip3Aa proteins. Larvae that were fed E-64 had increased mortality and prolonged developmental period, but these parameters were unaffected when larvae were fed Vip3Aa. The epithelial cells of midguts were intact and closely connected with the basal membrane when larvae were fed Vip3Aa, but the epithelial cells degenerated in the E-64 treatment. The activities of antioxidative enzymes and expression levels of detoxification-related genes in P. japonica larvae were not altered after exposure to Vip3Aa; however, these biochemical and molecular parameters were significantly changed in the E-64 treatment. The results demonstrate that Vip3Aa protein is not harmful to the predator P. japonica., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Propylea japonica is a very important predator in agricultural ecosystems, which could be exposed to Bt protein. In this study, the bacterial community of P. japonica fed with normal food and food containing Cry2Ab protein was characterized for the first time using qPCR and high-throughput sequencing approaches. Results showed no effect of Cry2Ab on P. japonica development and reproduction. The most abundant bacterial phylum was Firmicutes, and the most abundant genus was Staphylococcus. The total bacteria copy number was not significantly different across four larval stages. Bacteria species composition was gathered more closely in feed on sucrose solution (sucrose-fed) than in larvae only fed on pea aphid (aphid-fed), the diversity indices of some operational taxonomic unit (OTU) were significantly different between sucrose-fed and aphid-fed samples. Different instar larval stages of P. japonica fed with sucrose solution containing Cry2Ab Bt protein and found no effect on microbial community composition and total bacteria copy numbers. However, effects on relative abundance of microbes, copy numbers of Corynebacterium 1 and Glutamicibacter arilaitensis were observed significantly lower in Bt-fed first and fourth larval stages. Low and high concentrations of Cry2Ab protein altered the microbial abundance relative to sucrose-fed P. japonica and copy numbers of G. arilaitensis and Staphylococcus xylosus were significantly lower in Bt-fed samples than control sucrose-fed. Our results are the first report showing that feeding on Cry2Ab protein does not alter microbial species composition in P. japonica, but effects gene copy number of some dominant bacteria. Further investigations are needed to assess the effect of copy number change on P. japonica., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The transgenic rice line T1C-19 provides high resistance to lepidopteran pests because of the synthesis of the Bacillus thuringiensis (Bt) insecticidal protein Cry1C. It thus shows good prospect for commercial planting in China. Species of Cladocera, an order of aquatic arthropods commonly found in aquatic ecosystems such as rice paddies, might be exposed to the insecticidal protein released from Bt-transgenic rice-straw residues. For the study reported herein, we used Daphnia magna (water flea) as a representative of Cladocera to evaluate whether aquatic arthropods are adversely affected when exposed to Bt rice-straw leachates. We exposed D. magna to M4 medium containing various volume percentages of medium that had been incubated with T1C-19 rice straw or rice straw from its non-transformed near-isoline Minghui 63 (MH63) for 21 days. Compared with pure M4 medium (control), the fitness and developmental and reproduction parameters of D. magna decreased significantly when exposed to rice-straw leachates; conversely, no significant differences between the T1C-19 and MH63 rice-straw leachate treatments were observed, indicating that the Bt rice straw leachate did not adversely affect this non-target species., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In order to clarify the effects of soil salinity on the insect-resistance of boll in transgenic Bt cotton, potted plants of two Bt cotton cultivars Xinmian 33B (salt-sensitive) and Zhong 07 (salt-tolerant) were exposed to five levels of soil salinity (0, 0.15%, 0.30%, 0.45% and 0.60%). The results showed that Bt protein content of boll shell decreased with increasing soil salinity. Compared with the control (0% soil salinity level), the Bt protein content of boll shell decreased significantly when the soil salinity level was above 0.15% for Xinmian 33B and above 0.30% for Zhong 07. The reduction extent of Bt protein content of boll shell at 30 days post anthesis (DPA) was greater than that at 10 DPA under the same soil salinity level. Significant reductions of soluble protein contents, nitrate reductase (NR), and glutamate pyruvate transaminase (GPT) activities were observed when the boll shell Bt protein content was significantly reduced. The content of free amino acid, protease, and peptidase activity of boll shell significantly increased when the soil salinity level was above 0.30%. In conclusion, soil salinity affected boll shell nitrogen metabolism and reduced Bt protein synthesis. Middle and high soil salinity levels could enhance decomposition of Bt protein, which further decreases the expression level of insecticidal protein.

Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins produced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K 2 Cr 2 O 7 ), a known toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D. magna were not significantly different between Cry1C and negative control treatments but were significantly inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Pardosa pseudoannulata is a prevailing spider species, and has been regarded as an important bio-control agent of insect pests in farmland of China. However, the available genomic and transcriptomic databases of P. pseudoannulata and their venom are limited, which severely hampers functional genomic analysis of P. pseudoannulata. Recently high-throughput sequencing technology has been proved to be an efficient tool for profiling the transcriptome of relevant non-target organisms exposed to Bacillus thuringiensis (Bt) protein through food webs., Results: In this study, the transcriptome of the venom apparatus was analyzed. A total of 113,358 non-redundant unigenes were yielded, among which 34,041 unigenes with complete or various length encoding regions were assigned biological function annotations and annotated with gene ontology and karyotic orthologous group terms. In addition, 3726 unigenes involved in response to stimulus and 720 unigenes associated with immune-response pathways were identified. Furthermore, we investigated transcriptomic changes in the venom apparatus using tag-based DGE technique. A total of 1724 differentially expressed genes (DEGs) were detected, while 75 and 372 DEGs were functionally annotated with KEGG pathways and GO terms, respectively. qPCR analyses were performed to verify the DEGs directly or indirectly related to immune and stress responses, including genes encoding heat shock protein, toll-like receptor, GST and NADH dehydrogenase., Conclusion: This is the first study conducted to specifically investigate the venom apparatus of P. pseudoannulata in response to Bt protein exposure through tritrophic chain. A substantial fraction of transcript sequences was generated by high-throughput sequencing of the venom apparatus of P. pseudoannulata. Then a comparative transcriptome analysis showing a large number of candidate genes involved in immune response were identified by the tag-based DGE technology. This transcriptome dataset will provide a comprehensive sequence resource for furture molecular genetic research of the venom apparatus of P. pseudoannulata.

An insect resistance monitoring program was developed for Mexico to accommodate the commercial introduction and stewardship of Bt cotton. Between 1998 and 2015, field-collected geographic populations of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) were evaluated against Cry1Ac and Cry2Ab proteins of Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) to establish baseline susceptibility data before the commercial use of Bollgard (Cry1Ac) and Bollgard II (Cry1Ac and Cry2Ab) cotton. An annual monitoring program was subsequently established in which a single diagnostic concentration of each Bt protein was used in a diet overlay bioassay. The diagnostic concentration represented the concentration where larvae, evaluated in baseline studies, were reduced in weight by ≥97% relative to untreated controls or failed to molt to third instar after 5 d. In the monitoring study, populations were tested against Cry1Ac from 1998 through 2015, and against Cry2Ab from 2002 through 2004 and again from 2007 through 2015. None of the Cry1Ac-exposed larvae tested during the 18-yr period reached the third larval instar after an exposure of 5 d, and weight reduction relative to untreated control larvae was uniform at about 98-99%. For the 12 yr of Cry2Ab monitoring, no larvae reached third instar, and weight reduction was uniform at >97% relative to controls. These results indicate that H. zea susceptibility to Cry1Ac and Cry2Ab has not changed during the period Bollgard and Bollgard II have been cultivated in Mexico., (© The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America.)

Bollgard(®) III was developed by combining cotton events COT102 and MON 15985 through conventional breeding to improve efficacy against lepidopteran feeding damage. COT102 produces the Vip3Aa19 protein and MON 15985 produces the Cry1Ac and Cry2Ab2 proteins. COT102 × MON 15985 has also been bred with Roundup Ready Flex(®) cotton (MON 88913) that confers glyphosate tolerance. This study evaluated the activity of COT102 and MON 15985 and the combined activity of COT102 and MON 15985 against the cotton bollworm (CBW, Helicoverpa zea). COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 have comparable Vip3Aa19 and/or Cry1Ac, Cry2Ab2 protein expression levels as determined by enzyme-linked immunosorbent assay. CBW demonstrated concentration-dependent growth inhibition after 7-days of feeding on lyophilized leaf tissue derived from COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 incorporated into an artificial diet. Observed EC50 values for COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 were comparable (≤4% deviation) with the predicted EC50 value under the assumption of additivity using the combined activity of COT102 and MON 15985. No interaction in biological activity between COT102 and MON 15985 is consistent with results from competition and ligand blotting assays that demonstrated that Vip3Aa does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. The results from this study demonstrate that the activity of COT102 × MON 15985 against CBW is consistent with predictions of additivity., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, different Bacillus thuringiensis (Bt) toxin-encoding genes have been combined or 'stacked' in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA)., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

In fields of transgenic Bt rice, frogs are exposed to Bt proteins through consumption of both target and nontarget insects. In the present study, we assessed the risk posed by transgenic rice expressing a Cry1Ab/1Ac fusion protein (Huahui 1, HH1) on the development of Xenopus laevis. For 90 days, froglets were fed a diet with 30% HH1 rice, 30% parental rice (Minghui 63, MH63), or no rice as a control. Body weight and length were measured every 15 days. After sacrificing the froglets, we performed a range of biological, clinical, and pathological assessments. No significant differences were found in body weight (on day 90: 27.7 ± 2.17, 27.4 ± 2.40, and 27.9 ± 1.67 g for HH1, MH63, and control, respectively), body length (on day 90: 60.2 ± 1.55, 59.3 ± 2.33, and 59.7 ± 1.64 mm for HH1, MH63, and control, respectively), animal behavior, organ weight, liver and kidney function, or the microstructure of some tissues between the froglets fed on the HH1-containing diet and those fed on the MH63-containing or control diets. This indicates that frog development was not adversely affected by dietary intake of Cry1Ab/1Ac protein.