Greenhouse-Selected Resistance to Cry3Bb1-Producing Corn in Three Western Corn Rootworm Populations

Transgenic corn producing the Bacillus thuringiensis (Bt) toxin Cry3Bb1 has been useful for controlling western corn rootworm, Diabrotica virgifera virgifera LeConte, one of the most economically important crop pests in the United States. However, rapid evolution of resistance by this beetle to Bt corn producing Cry3Bb1 has been reported previously from the laboratory, greenhouse, and field. Here we selected in the greenhouse for resistance to Cry3Bb1 corn in three colonies of WCR derived from Kansas, Minnesota, and Wisconsin, respectively. Three generations of rearing on Cry3Bb1 corn significantly increased larval survival on Cry3Bb1 corn, resulting in similar survival in the greenhouse for selected colonies on Cry3Bb1 corn and isoline corn that does not produce Bt toxin. After four to seven generations of rearing on Cry3Bb1 corn, survival in the field on Cry3Bb1 corn relative to isoline corn more than doubled for selected colonies (72%) compared with control colonies (33%). For both selected and control colonies, survival in the field was significantly lower on Cry3Bb1 corn than on isoline corn. On isoline corn, most fitness components were similar for selected colonies and control colonies. However, fecundity was significantly lower for selected colonies than control colonies, indicating a fitness cost associated with resistance. The rapid evolution of resistance by western corn rootworm to Bt corn reported here and previously underlines the importance of effective resistance management for this pest.     

Gregarina coronata N. Sp. (Apicomplexa: Eugregarinida) Described from Adults of the Southern Corn Rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae)

ABSTRACT. Gregarina coronata n. sp. (Apicomplexa: Eugregarinida) is described from the adults of the Southern Corn Root Worm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae). Measurements given are means, in micrometers, taken from mature gamonts in association. Primite: protomerite hemi-ellipsoidal with basal tumidus, length 47.6, width 44.0, with cytoplasmic granule, apical crown apparent; deutomerite elongate ellipsoidal, length 227.9, width 81.3; epimerite absorbed into anterior in gamont, globular in trophozoite. Satellite: protomerite hemi-ellipsoidal, truncated at association interface to appear trapezoidal, length 39.2, width 49.6, with cytoplasmic granule; deutomerite elongate ellipsoidal, length 240.6, width 80.2; epimerite absorbed into anterior in gamont. Association caudofrontal and often precocious, occurring during growth of trophozoites. Gametocysts spherical, 115.3 in diameter, 132.9 with hyaline coat; producing multiple oocyst chains under moist storage in 24–36 h. Oocysts very uniform in shape and size, dorsad: doliform, length 6.4, equatorial width 3.4, polar width 2.9; pleuron: dorso-ventrally flattened, corpus concave with bicondylic termina; corpus height 0.98, width 4.44; terminus height 1.96, width 0.98.     

Insecticidal activity of Cry3Bb1 expressed in Bt maize on larvae of the Colorado potato beetle, Leptinotarsa decemlineata

Environmental risk assessment for genetically modified crops producing insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) includes the evaluation of adverse effects on non-target organisms. Although ELISA concentration measurements indicate the presence of Cry proteins, sensitive insect bioassays determine whether there is biological activity. The insecticidal activity of the coleopteran-active Cry3Bb1 expressed in different tissues of Bt maize, contained in maize-fed herbivores, and in spiked soil was measured in sensitive insect bioassays using larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Biological activity was confirmed of Cry3Bb1 contained in pulverized Bt maize pollen, roots, leaves, silk, and Bt maize-fed spider mites and western corn rootworm adults. When test substances were incorporated into artificial diet at the same concentrations of Cry3Bb1 (measured by ELISA), maize pollen and leaf litter exhibited lower toxicity than fresh plant material and maize-fed arthropods. This suggests that nutritional quality of food and degradation of Cry proteins may influence toxicity to insects. When soil was spiked with Cry3Bb1, the Bt protein was highly adsorbed and retained its full biological activity. Because toxicity of Cry proteins contained in different matrices cannot always be determined from ELISA values alone, sensitive insect bioassays can improve hazard and exposure assessments in environmental risk assessment of Bt crops.     

Secondary Contact and Admixture between Independently Invading Populations of the Western Corn Rootworm,Diabrotica virgifera virgifera in Europe

The western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is one of the most destructive pests of corn in North America and is currently invading Europe. The two major invasive outbreaks of rootworm in Europe have occurred, in North-West Italy and in Central and South-Eastern Europe. These two outbreaks originated from independent introductions from North America. Secondary contact probably occurred in North Italy between these two outbreaks, in 2008. We used 13 microsatellite markers to conduct a population genetics study, to demonstrate that this geographic contact resulted in a zone of admixture in the Italian region of Veneto. We show that i) genetic variation is greater in the contact zone than in the parental outbreaks; ii) several signs of admixture were detected in some Venetian samples, in a Bayesian analysis of the population structure and in an approximate Bayesian computation analysis of historical scenarios and, finally, iii) allelic frequency clines were observed at microsatellite loci. The contact between the invasive outbreaks in North-West Italy and Central and South-Eastern Europe resulted in a zone of admixture, with particular characteristics. The evolutionary implications of the existence of a zone of admixture in Northern Italy and their possible impact on the invasion success of the western corn rootworm are discussed.     

Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb,Cry3Ca,and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas, which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu⁴⁷ for the 70-kDa form or Ile⁸⁸ for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) from C. puncticollis larvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.     

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa,Cry1Ab,Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests,Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with ¹²⁵I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.     

Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens,Diatraea saccharalis and Spodoptera frugiperda

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC₅₀, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC₉₀, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC₉₀ level than at the LC₅₀ level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.     

Comparative effects of 1, 10-phenanthroline and 1, 7-phenanthroline on DNA and RNA synthesis in mouse spleen

When 1, 10-phenanthroline at 10^?4 mole/kg was administered intraperitoneally to C3H mice, a significant decrease of (³²P) Na₂HPO₄ incorporation into splenic DNA and RNA was noted within 15 min. The same dose or higher was required to significantly inhibit the incorporation of (5-³H) uridine and (methyl-³H) thymidine into splenic nucleic acid. 1, 10-phenanthroline also decrease the incorporation of the ³²P into DNA and RNA in 6C3HED ascites tumor. 1, 7-phenanthroline, a non-chelating analogue at 10^?4 mole/kg, less effectively altered the rate of the ³²P incorporation into splenic nucleic acid within 15 min, but significantly inhibited the incorporation within 1 hr.     

Establishment of Endoreticulatus N. G. for Pleistophora fidelis (Hostounský & Weiser, 1975) (Microsporida: Pleistophoridae) Based on the Ultrastructure of a Microsporidium in the Colorado Potato Beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)1,2

A new genus, Endoreticulatus n. g., is described for the microsporidium Pleistophora fidelis (Hostounský & Weiser, 1975) based on light and electron microscopic studies of a microsporidium in the Colorado potato beetle, Leptinotarsa decemlineata (Say). This latter microsporidium is considered to be conspecific with P. fidelis because both isolates have been shown to be infectious for L. decemlineata where infection is limited to the epithelial cells of the midgut; both are haplokaryotic and develop as groups of sporoblasts and spores in subpersistent vacuoles in the host cell. In addition, meronts, sporonts, and spores of each isolate often occur simultaneously in a common cell, and by light microscopy they both appear similar. Ultrastructural studies of the isolate from L. decemlineata revealed that all developmental stages occur in parasitophorous vacuoles derived from cisternae of rough endoplasmic reticulum of the host cell cytoplasm. Based on the unique nature of the parasitophorous vacuole, a new genus, Endoreticulatus, is proposed for P. fidelis. The genus is compared with the other genera whose species undergo multisporous sporogony in sporophorous vesicles. In addition, the nature of the parasitophorous vacuole of Endoreticulatus fidelis (Hostounský & Weiser, 1975) n. comb. is compared with the parasitophorous vacuole known to encase various developmental stages of several other microsporidian species.     

Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein,Functions in Cross-linking and Stabilizing Actin Filaments

Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments.     

Se14, Encoding a JmjC Domain-Containing Protein,Plays Key Roles in Long-Day Suppression of Rice Flowering through the Demethylation of H3K4me3 of RFT1

Floral transition from the vegetative to the reproductive growth phase is a major change in the plant life cycle and a key factor in reproductive success. In rice (Oryza sativa L.), a facultative short-day plant, numerous flowering time and flower formation genes that control floral transition have been identified and their physiological effects and biochemical functions have been clarified. In the present study, we used a Se14-deficient mutant line (HS112) and other flowering mutant lines to investigate the photoperiodic response, chromosomal location and function in the photoperiod sensitivity of the Se14 gene. We also studied the interactive effects of this locus with other crucial flowering time genes. We found that Se14 is independent of the known photoperiod-sensitive genes, such as Hd1 and Ghd7, and is identical to Os03g0151300, which encodes a Jumonji C (JmjC) domain-containing protein. Expression analysis revealed that the expressions of RFT1, a floral initiator known as a “florigen-like gene”, and Ehd1 were up-regulated in HS112, whereas this up-regulation was not observed in the original variety of ‘Gimbozu’. ChIP assays of the methylation states of histone H3 at lysine 4 (H3K4) revealed that the trimethylated H3K4 in the promoter region of the RFT1 chromatin was significantly increased in HS112. We conclude that Se14 is a novel photoperiod-sensitivity gene that has a suppressive effect on floral transition (flowering time) under long day-length conditions through the modification of chromatin structure by H3K4me3 demethylation in the promoter region of RFT1.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

RBEs of Thermal Neutron Capture Therapy and 10B(n,α)7 Li Reaction on Melanoma-Bearing Hamsters

Using Greene's melanoma transplanted into Syrian (golden) hamsters, we determined the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) using ¹⁰B-paraboronophenylalanine (¹⁰B-BPA) in comparison with a 9-MeV electron beam. We also obtained the RBE of the ¹⁰B(n,α)⁷Li reaction by calculation based on summed dose data from TNCT. Throughout this study, the Kyoto University Research Reactor was used as the source for thermal neutrons; the reactor was specially altered to attain a low contamination level both for gamma-rays and fast neutrons. ¹⁰B-BPA was administered 8 hours before thermal neutron irradiation to the hamsters with melanoma. The tumor was then irradiated at 5 MW for 90 minutes. The absorbed dose from this TNCT was calculated by the method of Fairchild and Goodman (Phys. Med. Biol. 1966; 2:15–30). The RBEs of the TNCT and the ¹⁰B(n,α)⁷Li reaction obtained by the tumor growth delay time (TGDT) method were 2.22 and 2.51, respectively, at 10.5 days of TGDT. These RBE values varied with TGDT and the absorbed dose. The RBE value of TNCT had a peak at 7.0 days of TGDT; that of the ¹⁰B(n,α)⁷Li reaction was higher at a low absorbed dose level and lower at a high absorbed dose level.     

Genetic Interactions between Reg1/Hex2 and Glc7, the Gene Encoding the Protein Phosphatase Type 1 Catalytic Subunit in Saccharomyces Cerevisiae

Mutations in GLC7, the gene encoding the type 1 protein phosphatase catalytic subunit, cause a variety of abberrant phenotypes in yeast, such as impaired glycogen synthesis and relief of glucose repression of the expression of some genes. Loss of function of the REG1/HEX2 gene, necessary for glucose repression of several genes, was found to suppress the glycogen-deficient phenotype of the glc7-1 allele. Deletion of REG1 in a wild-type background led to overaccumulation of glycogen as well as slow growth and an enlarged cell size. However, loss of REG1 did not suppress other phenotypes associated with GLC7 mutations, such as inability to sporulate or, in cells bearing the glc7(Y-170) allele, lack of growth at 14°. The effect of REG1 deletion on glycogen accumulation is not simply due to derepression of glucose-repressed genes, although it does require the presence of SNF1, which encodes a protein kinase essential for expression of glucose-repressed genes and for glycogen accumulation. We propose that REG1 has a role in controlling glycogen accumulation.     

Review: Biological Effectiveness of Thermal Neutrons and 10B(n,α)7Li Reaction on Cultured Cells

There are only a few reports on the relative biological effectiveness (RBE) of thermal neutrons and ¹⁰B(n,α)⁷Li reactions either in vitro or in vivo. The data in this paper summarize almost all previously published in vitro data. Because only a few reactors are available for biomedical purposes, it is difficult to make a comparison of data from experiments using the same kind of radiation, and also to make a comparison of data from experiments using the different kinds of radiations. However, it is indispensable for boron neutron capture therapy to make a radiobiological analysis. More intensive study, including repair process and oxygen effect, is necessary for establishing the fundamental basis of the clinical application of boron neutron capture therapy.