Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus).

The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.

Transcriptional sequencing and analysis of major genes involved in the adventitious root formation of mango cotyledon segments.

MAIN CONCLUSION: A total of 74,745 unigenes were generated and 1975 DEGs were identified. Candidate genes that may be involved in the adventitious root formation of mango cotyledon segment were revealed. Adventitious root formation is a crucial step in plant vegetative propagation, but the molecular mechanism of adventitious root formation remains unclear. Adventitious roots formed only at the proximal cut surface (PCS) of mango cotyledon segments, whereas no roots were formed on the opposite, distal cut surface (DCS). To identify the transcript abundance changes linked to adventitious root development, RNA was isolated from PCS and DCS at 0, 4 and 7 days after culture, respectively. Illumina sequencing of libraries generated from these samples yielded 62.36 Gb high-quality reads that were assembled into 74,745 unigenes with an average sequence length of 807 base pairs, and 33,252 of the assembled unigenes at least had homologs in one of the public databases. Comparative analysis of these transcriptome databases revealed that between the different time points at PCS there were 1966 differentially expressed genes (DEGs), while there were only 51 DEGs for the PCS vs. DCS when time-matched samples were compared. Of these DEGs, 1636 were assigned to gene ontology (GO) classes, the majority of that was involved in cellular processes, metabolic processes and single-organism processes. Candidate genes that may be involved in the adventitious root formation of mango cotyledon segment are predicted to encode polar auxin transport carriers, auxin-regulated proteins, cell wall remodeling enzymes and ethylene-related proteins. In order to validate RNA-sequencing results, we further analyzed the expression profiles of 20 genes by quantitative real-time PCR. This study expands the transcriptome information for Mangifera indica and identifies candidate genes involved in adventitious root formation in cotyledon segments of mango.

Annular Defects Impair the Mechanical Stability of the Intervertebral Disc.

STUDY DESIGN: A biomechanical study. OBJECTIVES: The purpose of this study was to investigate the effects of cruciform and square incisions of annulus fibrosus (AF) on the mechanical stability of bovine intervertebral disc (IVD) in multiple degrees of freedom. METHODS: Eight bovine caudal IVD motion segments (bone-disc-bone) were obtained from the local abattoir. Cruciform and square incisions were made at the right side of the specimen's annulus using a surgical scalpel. Biomechanical testing of three-dimensional 6 degrees of freedom was then performed on the bovine caudal motion segments using the mechanical testing and simulation (MTS) machine. Force, displacement, torque and angle were recorded synchronously by the MTS system. P value <.05 was considered statistically significant. RESULTS: Cruciform and square incisions of the AF reduced both axial compressive and torsional stiffness of the IVD and were significantly lower than those of the intact specimens (P < .01). Left-side axial torsional stiffness of the cruciform incision was significantly higher than a square incision (P < .01). Neither incision methods impacted flexional-extensional stiffness or lateral-bending stiffness. CONCLUSIONS: The cruciform and square incisions of the AF obviously reduced axial compression and axial rotation, but they did not change the flexion-extension and lateral-bending stiffness of the bovine caudal IVD. This mechanical study will be meaningful for the development of new approaches to AF repair and the rehabilitation of the patients after receiving discectomy.

Species variation in the cartilaginous endplate of the lumbar intervertebral disc.

Backgrounds: Cartilaginous endplate (CEP) plays an essential role in intervertebral disc (IVD) health and disease. The aim was to compare the CEP structure of lumbar IVD and to reveal the detailed pattern of integration between the CEP and bony endplate (BEP) from different species. Methods: A total of 34 IVDs (5 human, 5 goat, 8 pig, 8 rabbit, and 8 rat IVDs) were collected, fixed and midsagittally cut; in each IVD, one-half was used for histological staining to observe the CEP morphology, and the other half was used for scanning electron microscopy (SEM) analysis to measure the diameters and distributions of collagen fibers in the central and peripheral CEP areas and to observe the pattern of CEP-BEP integration from different species. Results: The human, pig, goat, and rabbit IVDs had the typical BEP-CEP structure, but the rat CEP was directly connected with the growth plate. Human CEP was the thickest (896.95 ± 87.71 µm) among these species, followed by pig, goat, rat, and rabbit CEPs. Additionally, the mean cellular density of the rabbit CEP was the highest, which was 930 ± 202 per mm2, followed by the rat, goat, pig, and human CEPs. In all the species, the collagen fiber diameter in the peripheral area was much bigger than that in the central area. The collagen fiber diameters of CEP from the human, pig, goat, and rat were distributed between 35 nm and 65 nm. The BEP and CEP were connected by the collagen from the CEP, aggregating into bundles or cross links with each other to form a network, and anchored to BEP. Conclusions: Significant differences in the thickness, cellular density, and collagen characterization of CEPs from different species were demonstrated; the integration of BEP-CEP in humans, pigs, goats, and rabbits was mainly achieved by the collagen bundles anchoring system, while the typical BEP-CEP interface did not exist in rats.

Over-expression of mango (Mangifera indica L.) MiARF2 inhibits root and hypocotyl growth of Arabidopsis.

An auxin response factor 2 gene, MiARF2, was cloned in our previous study [1] from the cotyledon section of mango (Mangifera indica L. cv. Zihua) during adventitious root formation, which shares an 84% amino acid sequence similarity to Arabidopsis ARF2. This study was to examine the effects of over-expression of the full-length MiARF2 open reading frame on the root and hypocotyl growth in Arabidopsis. Phenotype analysis showed that the T(3) transgenic lines had about 20-30% reduction in the length of hypocotyls and roots of the seedlings in comparison with the wild-type. The transcription levels of ANT and ARGOS genes which play a role in controlling organ size and cell proliferation in the transgenic seedlings also decreased. Therefore, the inhibited root and hypocotyl growth in the transgenic seedlings may be associated with the down-regulated transcription of ANT and ARGOS by the over-expression of MiARF2. This study also suggests that although MiARF2 only has a single DNA-binding domain (DBD), it can function as other ARF-like proteins containing complete DBD, middle region (MR) and carboxy-terminal dimerization domain (CTD).

Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms.

The application of recombinant DNA technology has resulted in many insect-resistant varieties by genetic engineering (GE). Crops expressing Cry toxins derived from Bacillus thuringiensis (Bt) have been planted worldwide, and are an effective tool for pest control. However, one ecological concern regarding the potential effects of insect-resistant GE plants on non-target organisms (NTOs) has been continually debated. In the present study, we briefly summarize the data regarding the development and commercial use of transgenic Bt varieties, elaborate on the procedure and methods for assessing the non-target effects of insect-resistant GE plants, and synthetically analyze the related research results, mostly those published between 2005 and 2010. A mass of laboratory and field studies have shown that the currently available Bt crops have no direct detrimental effects on NTOs due to their narrow spectrum of activity, and Bt crops are increasing the abundance of some beneficial insects and improving the natural control of specific pests. The use of Bt crops, such as Bt maize and Bt cotton, results in significant reductions of insecticide application and clear benefits on the environment and farmer health. Consequently, Bt crops can be a useful component of integrated pest management systems to protect the crop from targeted pests.

Reduced Mythimna separata infestation on Bt corn could benefit aphids.

Use of genetically engineered plants that express insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) have been proven efficacious for managing lepidopteran pests. However, in some cases herbivores that are not targeted by the Bt trait have increased in importance. It has been suggested that reduced caterpillar damage to Bt crops could lead to decreased levels of induced plant defensive compounds which might benefit other non-target herbivores. Here we investigated the potential effect of reduced damage by larvae of Mythimna separata on aphid populations in Bt corn. We compared the performance of Rhopalosiphum maidis feeding on non-Bt corn plants that had been infested by M. separata larvae or were uninfested. The results showed that caterpillar-infested corn plants significantly reduced the fitness of R. maidis leading to a prolonged nymphal development time, reduced adult longevity and fecundity compared to uninfested plants. Consequently, the population growth rate of corn aphids feeding on caterpillar-infested corn plants was significantly lower than on uninfested plants. As expected, the aphids performed significantly better on Lepidoptera-resistant Bt corn than on non-Bt corn when plants were infested with M. separata, since the caterpillars caused very little damage to the Bt plants. The current findings indicate that reduced M. separata infestation could benefit aphid development in Bt corn. Bt corn has the potential to be commercialized in China in the near future and aphids and other non-target pests should be monitored in the farming fields.

Modified surgical treatment for a patient with neurofibromatosis scoliosis: A case report.

BACKGROUND: The correction surgery for severely multidimensional spinal deformity in neurofibromatosis type I is very difficult and it is still a very big challenge for spine surgeons. CASE SUMMARY: A 44-year-old woman presented with progressive kyphosis for more than 10 years and low back pain for 2 years. She had been diagnosed with neurofibromatosis at a local hospital many years ago. Conservative treatments had been applied, but the symptoms got worse rather than alleviated. Therefore, surgery was required. CONCLUSION: For this patient with severe deformity, the correction treatment of Ponte osteotomy followed by satellite rod technique in the region of the apical vertebra and the technique of pedicle screws and dual iliac screws had been applied, and successful clinical outcomes were achieved.

Auxin efflux carriers, MiPINs, are involved in adventitious root formation of mango cotyledon segments.

Adventitious roots form only at the proximal cut surface (PCS) but not at the distal cut surface (DCS) of mango cotyledon segments. In this study, mango embryos treated with indole-3-butyric acid (IBA) showed significantly increased adventitious root formation, while those treated with 2, 3, 5-triiodobenzoic acid (TIBA) demonstrated complete inhibition of adventitious rooting. Mango embryos treated with auxin influx inhibitors demonstrated lower inhibition of adventitious roots than those treated with TIBA. The endogenous indol-3-acetic acid (IAA) content on the PCS and DCS was similar at 0 h, then increased on both surfaces after 6 h, and IAA content on the PCS were always higher than those on the DCS. We cloned three genes encoding auxin efflux carriers (i.e., MiPIN2-4) and examined their temporal and spatial expression patterns under different treatments. Relative expression of all MiPINs studied was very low at 0 h but significantly increased on both PCS and DCS from 1 d to 10 d, to varying degrees. We overexpressed MiPIN1-4 in Arabidopsis plants and found a significant increase in adventitious root quantity in MiPIN1 and MiPIN3 transgenic lines. Immunofluorescence results showed that MiPIN1 and MiPIN3 are primarily localized in the vascular tissues and the cells adjacent to abaxial surface. In conclusion, we propose that in mango cotyledon segments, wounding stimulates IAA biosynthesis, the transcription levels of PIN genes were significantly increased in different magnitudes on the PCS and DCS, resulting in polar IAA transport from the DCS to PCS via the vascular tissues, thereby triggering adventitious root formation.

Herbivore-induced rice resistance against rice blast mediated by salicylic acid.

In agro-ecosystems, plants are important mediators of interactions between their associated herbivorous insects and microbes, and any change in plants induced by one species may lead to cascading effects on interactions with other species. Often, such effects are regulated by phytohormones such as jasmonic acid (JA) and salicylic acid (SA). Here, we investigated the tripartite interactions among rice plants, three insect herbivores (Chilo suppressalis, Cnaphalocrocis medinalis or Nilaparvata lugens), and the causal agent of rice blast disease, the fungus Magnaporthe oryzae. We found that pre-infestation of rice by C. suppressalis or N. lugens but not by C. medinalis conferred resistance to M. oryzae. For C. suppressalis and N. lugens, insect infestation without fungal inoculation induced the accumulation of both JA and SA in rice leaves. In contrast, infestation by C. medinalis increased JA levels but reduced SA levels. The exogenous application of SA but not of JA conferred resistance against M. oryzae. These results suggest that pre-infestation by C. suppressalis or N. lugens conferred resistance against M. oryzae by increasing SA accumulation. These findings enhance our understanding of the interactions among rice plant, insects and pathogens, and provide valuable information for developing an ecologically sound strategy for controlling rice blast.

Safety Assessment of Bacillus thuringiensis Insecticidal Proteins Cry1C and Cry2A with a Zebrafish Embryotoxicity Test.

As a result of the large-scale planting of transgenic Bacillus thuringiensis (Bt) crops, fish would be exposed to freely soluble Bt insecticidal protein(s) that are released from Bt crop tissues into adjacent bodies of water or by way of direct feeding on deposited plant material. To assess the safety of two Bt proteins Cry1C and Cry2A to fish, we used zebrafish as a representative species and exposed their embryos to 0.1, 1, and 10 mg/L of the two Cry proteins until 132 h post-fertilization and then several developmental, biochemical, and molecular parameters were evaluated. Chlorpyrifos (CPF), a known toxicant to aquatic organisms, was used as a positive control. Although CPF exposure resulted in significant developmental, biochemical, and molecular changes in the zebrafish embryos, there were almost no significant differences after Cry1C or Cry2A exposure. Thus, we conclude that zebrafish embryos are not sensitive to Cry1C and Cry2A insecticidal proteins at test concentrations.

Ginkgolic acids induce HepG2 cell death via a combination of apoptosis, autophagy and the mitochondrial pathway.

Ginkgolic acids may induce malignant cell death via the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 apoptosis pathway. Concurrently, apoptosis, autophagy and mitochondrial dysfunction may also be involved in bringing about this endpoint. The anticancer effect of Ginkgolic acids (GAs) was investigated using the HepG2 cell line. The median lethal dose of the GAs of the HepG2 was measured via an MTT assay, the dose-response curves were evaluated and changes in cell morphology were monitored by microscopy. Autophagy in HepG2 cells was down regulated using 3-methyladenine (3-MA) or Beclin-1-specific small interfering RNA (siRNA) and the expression of apoptosis associated proteins caspase-3, Bax/Bcl-2, and the autophagy-associated protein 5 and microtubule-associated protein 1A/1B-light chain 3 in the GA-treated HepG2 cells were all measured by western blot analysis. The level of apoptosis in the GA-treated cells was also assessed using terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) assay, and the mitochondrial membrane potential (Δψm) was detected by immunofluorescence. The results of the MTT and TUNEL assays indicated that the proliferation of HepG2 cells treated with GAs was significantly reduced compared with the control group, and the rate of the inhibition was dose-dependent. Western blot analysis indicated that treatment with the Gas induced apoptosis and autophagy in the HepG2 cells. The Δψm of the GA-treated HepG2 cells was decreased compared with the control, as monitored by immunofluorescence. However, upon the administration of 3-MA or Beclin-1-specific siRNAs (inhibitors of the autophagy), the expression levels of the apoptosis- and autophagy-associated proteins were decreased. In conclusion, the results of the present study indicated that GAs are potent anticancer agents that function through a combination of the apoptosis, autophagy and mitochondrial pathways.

No effect of Bt Cry1Ie toxin on bacterial diversity in the midgut of the Chinese honey bees, Apis cerana cerana (Hymenoptera, Apidae).

Cry1Ie protein derived from Bacillus thuringiensis (Bt) has been proposed as a promising candidate for the development of a new Bt-maize variety to control maize pests in China. We studied the response of the midgut bacterial community of Apis cerana cerana to Cry1Ie toxin under laboratory conditions. Newly emerged bees were fed one of the following treatments for 15 and 30 days: three concentrations of Cry1Ie toxin (20 ng/mL, 200 ng/mL, and 20 µg/mL) in sugar syrup, pure sugar syrup as a negative control and 48 ng/mL imidacloprid as a positive control. The relative abundance of 16S rRNA genes was measured by Quantitative Polymerase Chain Reaction and no apparent differences were found among treatments for any of these counts at any time point. Furthermore, the midgut bacterial structure and compositions were determined using high-throughput sequencing targeting the V3-V4 regions of the 16S rDNA. All core honey bee intestinal bacterial genera such as Lactobacillus, Bifidobacterium, Snodgrassella, and Gilliamella were detected, and no significant changes were found in the species diversity and richness for any bacterial taxa among treatments at different time points. These results suggest that Cry1Ie toxin may not affect gut bacterial communities of Chinese honey bees.

Does Bt maize expressing Cry1Ac protein have adverse effects on the parasitoid Macrocentrus cingulum (Hymenoptera: Braconidae)?

The potential effects of insect-resistant, genetically engineered (GE) crops on non-target organisms, especially on predators and parasitoids, must be evaluated before their commercial cultivation. The effects of GE maize that produces Cry1Ac toxin on the parasitoid Macrocentrus cingulum were assessed by direct bioassay and indirect bioassay. In the indirect bioassay, parasitism rate, cocoon weight and the number of M. cingulum progeny produced per host were significantly reduced when M. cingulum-parasitized Cry1Ac-susceptible Ostrinia furnacalis were fed a diet containing purified Cry1Ac; however, life-table parameters of M. cingulum were not adversely affected when the same assay was performed with Cry1Ac-resistant O. furnacalis. These results indicated that the detrimental effects detected with a Cry1Ac-susceptible host were mediated by poor host quality. In a direct bioassay, no difference in life-table parameters were detected when M. cingulum adults were directly fed a 20% honey solution with or without Cry1Ac; however, survival and longevity were significantly reduced when M. cingulum adults were fed a honey solution containing potassium arsenate, which was used as a positive control. The stability and bioactivity of Cry1Ac toxin in the food sources and Cry1Ac toxin uptake by the host insect and parasitoid were confirmed by enzyme-linked immunosorbent assay and sensitive-insect bioassays. Our results demonstrate that M. cingulum is not sensitive to Cry1Ac toxin at concentrations exceeding those encountered in Bacillus thuringiensis maize fields. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms and will be useful for assessing other non-target impacts.

Molecular Cloning and Characterization of Four Genes Encoding Ethylene Receptors Associated with Pineapple (Ananas comosus L.) Flowering.

Exogenous ethylene, or ethephon, has been widely used to induce pineapple flowering, but the molecular mechanism behind ethephon induction is still unclear. In this study, we cloned four genes encoding ethylene receptors (designated AcERS1a, AcERS1b, AcETR2a, and AcETR2b). The 5' flanking sequences of these four genes were also cloned by self-formed adaptor PCR and SiteFinding-PCR, and a group of putative cis-acting elements was identified. Phylogenetic tree analysis indicated that AcERS1a, AcERS1b, AcETR2a, and AcETR2b belonged to the plant ERS1s and ETR2/EIN4-like groups. Quantitative real-time PCR showed that AcETR2a and AcETR2b (subfamily 2) were more sensitive to ethylene treatment compared with AcERS1a and AcERS1b (subfamily 1). The relative expression of AcERS1b, AcETR2a, and AcETR2b was significantly increased during the earlier period of pineapple inflorescence formation, especially at 1-9 days after ethylene treatment (DAET), whereas AcERS1a expression changed less than these three genes. In situ hybridization results showed that bract primordia (BP) and flower primordia (FP) appeared at 9 and 21 DAET, respectively, and flowers were formed at 37 DAET. AcERS1a, AcERS1b, AcETR2a, and AcETR2b were mainly expressed in the shoot apex at 1-4 DAET; thereafter, with the appearance of BP and FP, higher expression of these genes was found in these new structures. Finally, at 37 DAET, the expression of these genes was mainly focused in the flower but was also low in other structures. These findings indicate that these four ethylene receptor genes, especially AcERS1b, AcETR2a, and AcETR2b, play important roles during pineapple flowering induced by exogenous ethephon.

The effects of Bt Cry1Ie toxin on bacterial diversity in the midgut of Apis mellifera ligustica (Hymenoptera: Apidae).

The honey bee has been regarded as a key species in the environmental risk assessment of biotech crops. Here, the potential adverse effects of Cry1Ie toxin on the midgut bacteria of the worker bees (Apis mellifera ligustica) were investigated under laboratory conditions. Newly emerged bees were fed with different concentrations of Cry1Ie toxin syrups (20 ng/mL, 200 ng/mL, and 20 µg/mL), pure sugar syrup, and 48 ppb of imidacloprid syrups, then sampled after 15 and 30 d. We characterized the dominant midgut bacteria and compared the composition and structure of the midgut bacterial community in all samples using the Illumina MiSeq platform targeting the V3-V4 regions of 16S rDNA. No significant differences in the diversity of the midgut bacteria were observed between the five treatments. This work was the first to show the effects of Cry1Ie toxin on honey bees, and our study provided a theoretical basis for the biosafety assessment of transgenic Cry1Ie maize.

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis (Lepidoptera: Crambidae).

Two transgenic rice lines (T2A-1 and T1C-19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A-1 > Huahui 1 > T1C-19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C-19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C-19b providing an excellent performance at the maturing stage. By comparison, T1C-19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A-1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C-19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.

A 90 day safety assessment of genetically modified rice expressing Cry1Ab/1Ac protein using an aquatic animal model.

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.

Toxicological, Biochemical, and Histopathological Analyses Demonstrating That Cry1C and Cry2A Are Not Toxic to Larvae of the Honeybee, Apis mellifera.

The honey bee, Apis mellifera, is commonly used as a test species for the regulatory risk assessment of insect-resistant genetically engineered (IRGE) plants. In the current study, a dietary exposure assay was developed, validated, and used to assess the potential toxicity of Cry1C and Cry2A proteins from Bacillus thuringiensis (Bt) to A. mellifera larvae; Cry1C and Cry2A are produced by different IRGE crops. The assay, which uses the soybean trypsin inhibitor (SBTI) as a positive control and bovine serum albumin (BSA) as a negative control, was used to measure the responses of A. mellifera larvae to high concentrations of Cry1C and Cry2A. Survival was reduced and development was delayed when larvae were fed SBTI (1 mg/g diet) but were unaffected when larvae were fed BSA (400 µg/g), Cry1C (50 µg/g), or Cry2A (400 µg/g). The enzymatic activities of A. mellifera larvae were not altered and their midgut brush border membranes (BBMs) were not damaged after being fed with diets containing BSA, Cry1C, or Cry2A; however, enzymatic activities were increased and BBMs were damaged when diets contained SBTI. The study confirms that Cry1C and Cry2A have no acute toxicity to A. mellifera larvae at concentrations >10 times higher than those detected in pollen from Bt plants.

Tier-1 assays for assessing the toxicity of insecticidal proteins produced by genetically engineered plants to non-target arthropods.

In assessing an insect-resistant genetically engineered (IRGE) crop before its commercialization, researchers normally use so-called "Tier-1 assays" as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins (IPs) produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays: (i) methods for delivery of the IP to the test organisms; (ii) the need for and selection of compounds used as positive controls; and (iii) methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms.