Genetic diversity in pollen beetles (Meligethes aeneus) in Sweden: role of spatial,temporal and insecticide resistance factors

1 Pollen beetles Meligethes aeneus are pests of oilseed Brassica crops that are subject to intensive chemical control. Resistance to pyrethroids has been reported. Although this insect is of great economic importance, little is known about its genetic properties and population structure. 2 Amplified fragment length polymorphism (AFLP) analysis with the restriction endonuclease combination EcoRI and PstI was performed on 133 samples of groups of three pollen beetles collected during 2001–04 from five different provinces of Sweden. Both susceptible and resistant insects were studied. Using one primer combination, more than 450 polymorphic DNA fragments were obtained and, in total, four primer combinations were used for analysis. A subsample of 59 single beetles was analysed using one primer combination. 3 AFLP profiles were analysed by similarity measures using the Nei and Li coefficient and Neighbour‐joining dendrograms were generated. The dendrogram built using 133 samples showed three distinct groups, each containing beetles representing one generation. Statistical analysis using analysis of molecular variance of single beetle samples showed no evidence of significant genetic difference between resistant and susceptible beetles. Instead, a clear difference between samples, depending on time of collection and generation, was observed. 4 The expected regional population structure, although statistically significant, explained little of the variation. The levels of genetic variation within populations were very high. There appears to be a high rate of gene flow between pollen beetle populations. The implications of this in the context of insecticide resistance are discussed.     

Development of a synthetic plant volatile-based attracticide for female noctuid moths. III. Insecticides for adult Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

We investigated the efficacy of insecticides combined with a plant volatile-based attractant for Helicoverpa armigera moths, under laboratory and field conditions. In the laboratory, 16 insecticides were assessed by the level of mortality and time to incapacitate and kill moths. The proboscis extension reflex technique was used for dosing moths. The pyrethroids, bifenthrin (only when synergised by the addition of piperonyl butoxide (PBO) but not without it) and cyfluthrin (with or without PBO), endosulfan, the carbamates methomyl and thiodicarb, and spinosad killed all moths tested at rates equivalent to, or less than, those which would be applied in cover sprays targeting larvae. The shortest time to moth incapacitation and death was observed with methomyl and thiodicarb. Spinosad produced very high mortality but moths took much longer to die. The two pyrethroids gave relatively slow kills, as did endosulfan. In a field trial, four insecticides were combined with the attractant and dead moths were collected daily from 1 to 4 days after application of the attracticide on 50 m rows of cotton. Significantly more dead moths ( H. armigera , H. punctigera and other noctuids) were found near the rows treated with attracticide containing methomyl compared with spinosad, fipronil and deltamethrin. For determining the impact of attracticides by recovering dead moths, quick acting insecticides are required to prevent moths flying away from the treated area to locations where they cannot be found. Methomyl and thiodicarb are suitable for this, but other insecticides especially spinosad could be used where quick action is not needed. Large numbers of moths were killed in the field trial, suggesting that attracticides for female Helicoverpa spp. moths could have significant impacts on local populations of these pests.     

Cloning,recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly,Lucilia cuprina

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non‐specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC₅₀ values at µg levels. Trimethoprim and pyrimethamine were 5–30‐fold less active. Relative IC₅₀ values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active‐site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.     

Does behaviour play a role in house fly resistance to imidacloprid‐containing baits?

The objective of this research was to examine the role and type of behavioural mechanisms that function in house fly, Musca domestica L. (Diptera: Muscidae), resistance to an imidacloprid‐containing commercial fly bait, QuickBayt^®, using an insecticide‐susceptible and an imidacloprid‐resistant strain. Mortality and feeding behaviour were observed through choice bioassays of three post‐imidacloprid selected house fly generations to determine whether flies would consume the bait in the presence of an alternative food source. Mortality rates in choice containers progressively decreased in post‐selection flies as QuickBayt^® no‐choice selections proceeded. There were no differences between the proportions of flies observed contacting QuickBayt^® and sugar, respectively, a finding that eliminates repellency as a mechanism of stimulus‐dependent behavioural resistance. However, differences in QuickBayt^® consumption and subsequent mortality between choice and no‐choice containers provided strong support for the evolution of consumption irritancy‐ or taste aversion‐related behavioural resistance. The results of this study support the responsible rotation of insecticide bait formulations for house fly control.     

Recognition and avoidance of insecticide-treated Scots Pine (Pinus sylvestris) by Hylobius abietis (Coleoptera: Curculionidae): implications for pest management strategies

Abstract 1 The feeding preferences of Hylobius abietis (L.) were studied in a series of choice and no‐choice trials for insecticide‐treated food, time‐to‐death studies and arena trials. 2 Treatment of Scots Pine twigs with a pyrethroid insecticide, lambda‐cyhalothrin, was compared with twigs treated with imidacloprid, a neonicotinoid. 3 Clear avoidance of insecticide‐treated food sources, with strong evidence of selection for untreated food sources, was shown. 4 In addition, it took up to 3 weeks for H. abietis to die from insecticide poisoning when fed on treated food and, during this time, it was potentially capable of finding new untreated food sources.     

Ecdysteroid resistant subclones of the epithelial cell line from Chironomus Tentans (insecta, diptera). I. Selection and characterization of resistant clones

Summary Chironomus tentans cells were cultured in the presence of gradually increasing concentrations of 20-OH-ecdysone or a nonsteroidal molting hormone agonist, the benzoylhydrazine RH 5992, for a period of about 2 yr. From these cultures, subclones were selected, which are resistant to up to 25 μM 20-OH-ecdysone according to morphological (changes in cell shape and cell arrangement) and physiological criteria (acetylcholinesterase induction, secretion of chitinolytic enzymes, thymidine incorporation). Some subclones, selected in the presence of 20-OH-ecdysone, are resistant only to molting hormone, but still respond to RH 5992 morphologically and biochemically, whereas subclones selected in the presence of the benzoylhydrazine showed no reaction neither to 20-OH-ecdysone nor to the hormone agonist. Hormone resistance is stable; 3 mo. after hormone withdrawal, resistant clones still do not respond to renewed exposure to 20-OH-ecdysone or RH 5992, respectively. Because in all resistant subclones tested so far all hormonally regulated responses known from sensitive cells were no longer detectable, it is assumed that the hormone signaling pathway itself is interrupted. Possible mechanisms of hormone resistance were discussed.     

Lethal and sub‐lethal effects of faecal deltamethrin residues on dung‐feeding insects

Endectocides administered to livestock to facilitate pest and parasite control may be excreted in the faeces at concentrations that are toxic to coprophagous insects, including species of ecological importance. Although much research has focused on the effects of macrocyclic lactones, relatively less attention has been given to any similar impacts of the widely used pyrethroid insecticides. Here, the effects of faecal residues of the pyrethroid deltamethrin after application to Holstein–Friesian cattle in a proprietary pour‐on formulation are examined. Freshly dropped dung was collected 1, 3, 5 and 7 days after treatment and from an untreated control group. In laboratory bioasssays, female Lucilia sericata (Diptera: Calliphoridae) blow flies matured significantly smaller egg batches and had a lower percentage of eggs hatch after feeding on dung collected for up to 5 days after treatment, compared with flies feeding on dung from untreated cattle. In the field, artificial dung pats were constructed from the collected dung and left on pastureland for 7 days before being retrieved and searched for insects. Significantly more adult Diptera emerged from the faeces of untreated cattle than from the dung of treated cattle collected on days 1 and 3 after treatment. Adult Coleoptera were found in lower numbers in the dung of treated animals compared with control dung, suggesting a repellent effect. The results indicate that deltamethrin residues in cattle faeces have a range of lethal and sub‐lethal effects on dung‐feeding insects for up to a week after treatment, but that the precise duration and nature of toxicity varies depending on the sensitivity of the insect in question.     

Genetic analysis of resistance to azinphosmethyl in the pear psylla Cacopsylla pyri

Laboratory selection with azinphosmethyl had little effect on the chemical resistance in Cacopsylla pyri strains, in comparison to the original wild populations. The resistance ratio relative to a susceptible strain varied from 10- to 40-fold depending on the generation studied. Crosses between two resistant strains and the susceptible strain show resistance to be autosomally inherited and semi-dominant in expression. Backcrosses between F1 and the susceptible strain were unable to distinguish unabigously between monogenic and polygenic inheritance. In the majority of experiments, however, the overall dose-response relationship for backcross progeny was consistent with a single gene hypothesis. Additional bioassays showed azinphosmethyl-resistant strain to cross-resistant monocrotophos and phosmet, but not carbamates, pyrethroids, amitraz or the organophosphates chlorpyrifos and mevinphos.     

Testing Anopheles albimanus for genetic linkage of insecticide resistance genes by combining insecticide bioassay and biochemical methods

A microtitre-plate assay which distinguishes propoxur-resistant from susceptibles Anopheles albimanus Weidemann was used to test for linkage between the genes for propoxur- and dieldrin-resistance. The adult progeny of a backcross between a doubly-resistant colony and a fully susceptible colony were exposed in conventional test kits to the standard discriminating dose of dieldrin, and kept in the insectary overnight. Both live and dead insects were then assayed individually for propoxur-resistance. The results showed that heterozygotes for propoxur-resistance could be reliably distinguished from susceptibles whether or not they had been killed up to 24 h previously by dieldrin treatment. In this way all the backcross progeny could be scored at both resistance loci, and all four genotypic classes identified. Resistant and susceptible alleles at the two loci were inherited independently, demonstrating the absence of linkage. The usual method of testing for linkage between resistance genes is inefficient and open to bias, because insects have to be exposed to each insecticide in turn, and only half of them can be scored at both loci. The method shown here avoids these drawbacks.