Novel Chitinolytic Enzymes with Biological Activity Against Herbivorous Insects

The soil bacteria, Streptomyces albidoflavus, secretes endochitinases and chitobiosidases that are active over a broad range of pH (4-10). Ingestion of this mixture of chitinolytic enzymes significantly reduced the growth and development of Trichoplusia ni and significantly reduced survival of Myzus persicae, Bemisia argentifolii, and Hypothenemus hampei. Perfusion chromatography was used to separate endochitinases from chitobiosidases. The endochitinases had significantly greater biological activity against Bemisia argentifolii than the chitobiosidases. The utility of chitinolytic enzymes as regulators of populations of herbivorous insects is discusse

Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac Is Independent of Alteration of the Cadherin-Like Receptor for Cry Toxins

Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin alteration

Seasonal Changes in <i>Thrips tabaci</i> Population Structure in Two Cultivated Hosts

<div><p><i>Thrips tabaci</i> is a major pest of high-value vegetable crops and understanding its population genetics will advance our knowledge about its ecology and management. Mitochondrial cytochrome oxidase subunit I (COI) gene sequence was used as a molecular marker to analyze <i>T. tabaci</i> populations from onion and cabbage fields in New York. Eight COI haplotypes were identified in 565 <i>T. tabaci</i> individuals collected from these fields. All <i>T. tabaci</i> were thelytokous and genetically similar to those originating from hosts representing seven plant families spanning five continents. The most dominant haplotype was NY-HT1, accounting for 92 and 88% of the total individuals collected from onion fields in mid-summer in 2005 and 2007, respectively, and 100 and 96% of the total in early fall in 2005 and 2007, respectively. In contrast, <i>T. tabaci</i> collected from cabbage fields showed a dynamic change in population structure from mid-summer to early fall. In mid-summer, haplotype NY-HT2 was highly abundant, accounting for 58 and 52% of the total in 2005 and 2007, respectively, but in early fall it decreased drastically to 15 and 7% of the total in 2005 and 2007, respectively. Haplotype NY-HT1 accounted for 12 and 46% of the total in cabbage fields in mid-summer of 2005 and 2007, respectively, but became the dominant haplotype in early fall accounting for 81 and 66% of the total in 2005 and 2007, respectively. Despite the relative proximity of onion and cabbage fields in the western New York landscape, <i>T. tabaci</i> populations differed seasonally within each cropping system. Differences may have been attributed to better establishment of certain genotypes on specific hosts or differing colonization patterns within these cropping systems. Future studies investigating temporal changes in <i>T. tabaci</i> populations on their major hosts in these ecosystems are needed to better understand host-plant utilization and implications for population management.</p></div

Inheritance of Resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F(1) reciprocal crosses and backcrosses between F(1) larvae and resistant (P(R)) and susceptible (P(S)) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F(1) crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F(1) larvae was slightly higher than that for P(S), suggesting that resistance is partially recessive. The responses of both backcross progeny (F(1) × P(R), F(1) × P(S)) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F(1) × P(R) backcross but decreased the correspondence with the F(1) × P(S) backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene

Relative levels of cadherin mRNA, normalized to the ß-actin mRNA, in the midgut of the susceptible and Cry1Ac resistant larvae determined by real-time RT-PCR analysis.

<p>Error bars indicate standard errors of the means from analysis of 3 individuals.</p

The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant <i>T. ni</i> by toxin overlay binding analysis.

<p>The 200 kDa Cry1Ac-binding cadherin was detected in midgut BBMV proteins from both susceptible and resistant <i>T. ni</i> by toxin overlay binding analysis.</p

Cadherin allele frequencies in F₂ progenies from four single-pair cross families.

1<p>Statistical significance was analyzed by Chi-square test with the predicted ratios of <i>cad^Ccad^C</i> : <i>cad^Ccad^G</i> : <i>cad^Gcad^G</i>  = 1∶2∶1 in the tested individuals of each treatment.</p

Response of F₂ larvae from four families of single-pair crosses to Cry1Ac selection.

1<p>Survival Rate (%) was corrected using the Abbott’s formula <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035991#pone.0035991-Abbott1" target="_blank">[63]</a> with the control survival rates which were ≥95% in the bioassays.</p>2<p>Predicted survival rate 25% to selection with Cry1Ac was calculated based on inheritance of the recessive monogenic Cry1Ac-resistance trait.</p>3<p>Statistical significance was tested by <i>chi</i>-square test.</p

Mechanism of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Greenhouse Population of the Cabbage Looper, Trichoplusia ni

The cabbage looper, Trichoplusia ni, is one of only two insect species that have evolved resistance to Bacillus thuringiensis in agricultural situations. The trait of resistance to B. thuringiensis toxin Cry1Ac from a greenhouse-evolved resistant population of T. ni was introgressed into a highly inbred susceptible laboratory strain. The resulting introgression strain, GLEN-Cry1Ac-BCS, and its nearly isogenic susceptible strain were subjected to comparative genetic and biochemical studies to determine the mechanism of resistance. Results showed that midgut proteases, hemolymph melanization activity, and midgut esterase were not altered in the GLEN-Cry1Ac-BCS strain. The pattern of cross-resistance of the GLEN-Cry1Ac-BCS strain to 11 B. thuringiensis Cry toxins showed a correlation of the resistance with the Cry1Ab/Cry1Ac binding site in T. ni. This cross-resistance pattern is different from that found in a previously reported laboratory-selected Cry1Ab-resistant T. ni strain, evidently indicating that the greenhouse-evolved resistance involves a mechanism different from the laboratory-selected resistance. Determination of specific binding of B. thuringiensis toxins Cry1Ab and Cry1Ac to the midgut brush border membranes confirmed the loss of midgut binding to Cry1Ab and Cry1Ac in the resistant larvae. The loss of midgut binding to Cry1Ab/Cry1Ac is inherited as a recessive trait, which is consistent with the recessive inheritance of Cry1Ab/Cry1Ac resistance in this greenhouse-derived T. ni population. Therefore, it is concluded that the mechanism for the greenhouse-evolved Cry1Ac resistance in T. ni is an alteration affecting the binding of Cry1Ab and Cry1Ac to the Cry1Ab/Cry1Ac binding site in the midgut