Identification of factors responsible for insecticide resistance in Helicoverpa armigera

Moth larvae (Helicoverpa armigera Hübner) collected from field crops were tested for resistance to cypermethrin, fenvalerate, endosulfan, monocrotophos and quinolphos. Larvae were treated with a dose of the pesticide that would kill 99% of the susceptible insects. The percent survival of the resistant strains was determined. Highest seasonal average percentage survival was recorded by fenvalerate (65.0%) followed by cypermethrin (62.4%). Acetylcholinesterase of resistant larvae was less sensitive to monocrotophos and methyl paraoxon. Resistant larvae showed higher activities of esterases, phosphatases and methyl paraoxon hydrolase compared with susceptible larvae. The presence of high activity of esterases was attributed to appearance of extra bands of esterases in native PAGE. The presence of P-glycoprotein expression was detected in resistant larvae using P-gp antibodies; this was not detected in the susceptible larvae. Our results indicate that the high level of resistance detected in the field pests could be because of a combined effect of decreased sensitivity to AChE, higher levels of esterases, phosphatases and the expression of P-gp.     

Structural conservation in the major facilitator superfamily as revealed by comparative modeling

The structures of membrane transporters are still mostly unsolved. Only recently, the first two high-resolution structures of transporters of the major facilitator superfamily (MFS) were published. Despite the low sequence similarity of the two proteins involved, lactose permease and glycerol-3-phosphate transporter, the reported structures are highly similar. This leads to the hypothesis that all members of the MFS share a similar structure, regardless of their low sequence identity. To test this hypothesis, we generated models of two other members of the MFS, the Tn10-encoded metal-tetracycline/H(+) antiporter (TetAB) and the rat vesicular monoamine transporter (rVMAT2). The models are based on the two MFS structures and on experimental data. The models for both proteins are in good agreement with the data available and support the notion of a shared fold for all MFS proteins.     

Peanut Stripe Potyvirus Resistance in Peanut (Arachis Hypogaea L.) Plants Carrying Viral Coat Protein Gene Sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.     

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

Between‐year variation of MHC allele frequencies in great reed warblers: selection or drift?

The major histocompatibility complex (MHC) genes are extremely polymorphic and this variation is assumed to be maintained by balancing selection. Cyclic interactions between pathogens and their hosts could generate such selection, and specific MHC alleles or heterozygosity at certain MHC loci have been shown to confer resistance against particular pathogens. Here we compare the temporal variation in allele frequencies of 23 MHC class I alleles with that of 23 neutral microsatellite markers in adult great reed warblers (a passerine bird) in nine successive cohorts. Overall, the MHC alleles showed a significantly higher variation in allele frequencies between cohorts than the microsatellite alleles, using a multi-variate genetic analysis (amova). The frequency of two specific MHC alleles, A3e (P = 0.046) and B4b (P = 0.0018), varied more between cohorts than expected from random, whereas none of the microsatellite alleles showed fluctuations exceeding the expectation from stochastic variation. These results imply that the variation in MHC allele frequencies between cohorts is not a result of demographic events, but rather an effect of selection favouring different MHC alleles in different years.     

Characterization of cultured insect cells selected by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> crystal toxin

Summary Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hübner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1, 294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-d-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expression. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.     

Resistance to macrolide, lincosamide, streptogramin, ketolide, and oxazolidinone antibiotics

Macrolides have enjoyed a resurgence as new derivatives and related compounds have come to market. These newer compounds have become important in the treatment of community-acquired pneumoniae and nontuberculosis-Mycobacterium diseases. In this review, the bacterial mechanisms of resistance to the macrolide, lincosamide, streptogramin, ketolide, and oxazolidinone antibiotics, the distribution of the various acquired genes that confer resistance, as well as mutations that have been identified in clinical and laboratory strains are examined.     

The acrAB locus is involved in modulating intracellular acetyl coenzyme A levels in a strain of Escherichia coli CM2555 expressing the chloramphenicol acetyltransferase (cat) gene

Recently, an Escherichia coli CM2555 strain was described as sensitive to chloramphenicol when expressing the chloramphenicol resistance gene (cat) from a multicopy plasmid. This sensitivity was linked to dysfunction of the acrA gene, which encodes a component of the AcrAB-TolC multidrug efflux pump. Preliminary data indicate that the sensitivity phenotype might be due to a decline in intracellular acetyl coenzyme A concentration accompanying the reaction catalyzed by chloramphenicol acetyltransferase, the cat-encoded resistance protein. Here, we demonstrate that the acrA dysfunction is the factor impairing the intracellular acetyl coenzyme A levels in the cat-expressing CM2555 strain. This effect might be alleviated by the interplay of proteins constituting two homologous efflux systems: AcrAB-TolC and AcrEF-TolC. However, our results show also that this is a genetic background-specific phenomenon, as the decrease in acetyl coenzyme A level is not evident in a cat-bearing acrAB derivative of the commonly used strain C600.     

Pea Lectin Expressed Transgenically in Oilseed Rape Reduces Growth Rate of Pollen Beetle Larvae

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T₀-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T₂-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T₂-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.     

Heritability of resistance against ectoparasitism in the Drosophila-Macrocheles system

Ectoparasites are abundant in natural communities, can have pronounced deleterious fitness consequences to their host and are important vectors of transmissible parasitic disease. Yet very few studies have estimated the magnitude of heritable genetic variation underlying resistance against ectoparasitism, which significantly limits our ability to predict the evolution of this ecologically important character. The present paper reports results of artificial selection for increased resistance in Drosophila nigrospiracula against ectoparasitic, haematophagous mites, Macrocheles subbadius. In this system, which occurs naturally in the Sonoran Desert of North America, ectoparasitism significantly damages the expression of host fitness traits, including longevity, fecundity and male mating success. In the present study, resistance, which was modelled as a threshold trait, responded significantly to selection applied on either sex. Realized heritability, calculated as a mean across four replicates, was estimated to be 0.152 +/- 0.014 (SE). The heritability estimate from selection on males did not differ from that on females, but both estimates differed significantly from zero. This documented presence of additive genetic variation for resistance, coupled with knowledge of the fitness consequences of ectoparasitism, indicates that the host population possesses significant evolutionary potential. Selection was applied on the pre-attachment phase, thereby targeting behavioural forms of defence. This study therefore establishes parallels between insects and other animals in their ability to protect themselves and evolve behavioural defences against ectoparasites.