Jeffrey G. Scott

Cytochromes P450 and insecticide resistance

The cytochrome P450-dependent monooxygenases (monooxygenases) are an extremely important metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Monooxygenase-mediated metabolism is a common mechanism by which insects become resistant to insecticides as evidenced by the numerous insect species and insecticides affected. This review begins by presenting background information about P450s, the role of monooxygenases in insects, and the different techniques that have been used to isolate individual insect P450s. Next, insecticide resistance is briefly described, and then historical information about monooxygenase-mediated insecticide resistance is reviewed. For any case of monooxygenase-mediated resistance, identification of the P450(s) involved, out of the dozens that are present in an insect, has proven very challenging. Therefore, the next section of the review focuses on the minimal criteria for establishing that a P450 is involved in resistance. This is followed by a comprehensive examination of the literature concerning the individual P450s that have been isolated from insecticide resistant strains. In each case, the history of the strain and the evidence for monooxygenase-mediated resistance are reviewed. The isolation and characterization of the P450(s) from the strain are then described, and the evidence of whether or not the isolated P450(s) is involved in resistance is summarized. The remainder of the review summarizes our current knowledge of the molecular basis of monooxygenase-mediated resistance and the implications for the future. The importance of these studies for development of effective insecticide resistance management strategies is discussed.

Towards the elements of successful insect RNAi

RNA interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that the efficiency of RNAi varies between different species, the mode of RNAi delivery, and the genes being targeted. There is also variation in the duration of transcript suppression. At present, we have a limited capacity to predict the ideal experimental strategy for RNAi of a particular gene/insect because of our incomplete understanding of whether and how the RNAi signal is amplified and spread among insect cells. Consequently, development of the optimal RNAi protocols is a highly empirical process. This limitation can be relieved by systematic analysis of the molecular physiological basis of RNAi mechanisms in insects. An enhanced conceptual understanding of RNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application of RNAi to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods, particularly the honey bee (Apis mellifera) and cultured Pacific white shrimp (Litopenaeus vannamei) from viral and parasitic diseases.

Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins.

In the last decade, studies of individual insect P450s have blossomed. This new information has furthered our understanding of P450 diversity, insecticide resistance and tolerance to plant toxins. Insect P450s can be adult specific, larval specific or life stage independent. Similarly, insect P450s vary as to the tissues where they are expressed and in their response to inducers. Insect P450s can now be rapidly sequenced using degenerate PCR primers. Given the huge diversity represented by the Class Insecta, this technique will provide vast amounts of new information about insect P450s and the evolution of the P450 gene superfamily. CYP6D1 is responsible for monooxygenase-mediated resistance to pyrethroid insecticides in the house fly. CYP6D1 is ubiquitously expressed in adults with 10-fold higher levels found in the resistant strain compared to susceptible strains. CYP6D1 is on autosome 1 in house fly. The high level of expression found in the resistant strain is due to genes on autosomes 1 and 2. Whether or not the different CYP6D1 alleles found in resistant and susceptible strains have any role in resistance remains to be elucidated. The CYP6B gene subfamily is involved in the metabolism of host plant toxins (i.e. furanocoumarins). CYP6B gene transcripts in two Papilio (swallowtail) species have been shown to be induced by host plant toxins and in turn to metabolize these toxins. CYP6B P450s play a critical role in allowing Papilio to adapt to furanocoumarin-containing host plants. Similarities in structural and promoter regions of the CYP6B genes suggest that they are derived from a common ancestral gene. Although the P450 monooxygenases of insects are important for the metabolism of hormones and phermones, no individual P450 has yet been shown to metabolize an endogenous compound. Advances in this area are critical because they will provide important new information about insect physiology, biochemistry and development.

Increased transcription of CYP6D1 causes cytochrome P450-mediated insecticide resistance in house fly

Insecticide resistance is a major problem that continues to plague efforts to control pests of animals and crops. An important mechanism by which insects become resistant to insecticides is via increased detoxification mediated by the cytochrome P450 microsomal monooxygenases (monooxygenases). One of the fundamental gaps in our knowledge about this resistance mechanism is an understanding of how insects express high levels of the specific cytochrome P450(s) responsible for resistance. One such P450, CYP6D1, causes resistance to pyrethroid in the house fly and is expressed at 9-fold higher levels (mRNA and protein) in the Learn Pyrethroid Resistant (LPR) strain (compared to susceptible strains). The relative stability of CYP6D1 mRNA in resistant and susceptible strains was measured following inhibition of transcription with actinomycin D. The same time course of decrease in CYP6D1 mRNA abundance was detected in both strains indicating that the high level of expression of CYP6D1 in LPR is not due to increased stability of the mRNA. The comparative rates of transcription of CYP6D1 were measured using an in vitro run-on transcription assay. The relative amount of CYP6D1 transcript produced in this assay was 10-fold greater in the LPR strain compared to the susceptible strain. This demonstrates that increased transcription of CYP6D1 is an underlying cause of monooxygenase-mediated insecticide resistance. The increased rate of transcription of CYP6D1 in the resistant strain (LPR) is controlled by two factors: one on autosome 1 and another on autosome 2.

Insecticide Susceptibility of Aedes aegypti and Aedes albopictus across Thailand

Abstract Aedes aegypti (L.) and Aedes albopictus (Skuse), two important vectors of dengue fever and dengue hemorrhagic fever, were collected from Mae Sot, Nakhon Sawan, Nakhon Ratchasima, Surat Thani, and Phatthalung, Thailand, from July 2003 to April 2004. The patterns of insecticide susceptibility to temephos, malathion, and permethrin of both Ae. aegypti and Ae. albopictus larvae were determined. Ae. aegypti from all study sites were resistant to permethrin, they but were susceptible to malathion. Resistance to temephos was detected in all strains of Ae. aegypti, except those from Nakhon Ratchasima. Ae. albopictus larvae had low levels of resistance to all three insecticides, except Mae Sot and Phatthalung strains, which were resistant to permethrin.

Spinosad resistance in the housefly, Musca domestica, is due to a recessive factor on autosome 1

Abstract Spinosad is a new and highly promising insecticide with efficacy against a wide range of insects, including houseflies. Selection of the field collected houseflies produced a highly spinosad resistant (>150-fold) strain of housefly following 10 generations of selection. Spinosad resistance was a recessive trait linked to autosome 1 which could not be overcome with the insecticide synergists piperonyl butoxide, S , S , S -tributylphosphorotrithioate nor diethyl maleate. Selection for resistance to spinosad did not result in cross-resistance to other insecticides. These results suggest spinosad resistance in the housefly is due to a unique resistance mechanism.

Genome of the house fly, Musca domestica L., a global vector of diseases with adaptations to a septic environment

BackgroundAdult house flies, Musca domestica L., are mechanical vectors of more than 100 devastating diseases that have severe consequences for human and animal health. House fly larvae play a vital role as decomposers of animal wastes, and thus live in intimate association with many animal pathogens.ResultsWe have sequenced and analyzed the genome of the house fly using DNA from female flies. The sequenced genome is 691 Mb. Compared with Drosophila melanogaster, the genome contains a rich resource of shared and novel protein coding genes, a significantly higher amount of repetitive elements, and substantial increases in copy number and diversity of both the recognition and effector components of the immune system, consistent with life in a pathogen-rich environment. There are 146 P450 genes, plus 11 pseudogenes, in M. domestica, representing a significant increase relative to D. melanogaster and suggesting the presence of enhanced detoxification in house flies. Relative to D. melanogaster, M. domestica has also evolved an expanded repertoire of chemoreceptors and odorant binding proteins, many associated with gustation.ConclusionsThis represents the first genome sequence of an insect that lives in intimate association with abundant animal pathogens. The house fly genome provides a rich resource for enabling work on innovative methods of insect control, for understanding the mechanisms of insecticide resistance, genetic adaptation to high pathogen loads, and for exploring the basic biology of this important pest. The genome of this species will also serve as a close out-group to Drosophila in comparative genomic studies.

Evaluation of Novel Insecticides for Control of Dengue Vector Aedes aegypti (Diptera: Culicidae)

Abstract Insecticides are one of the major tools for controlling vector populations and for reducing the transmission of human pathogens. However, there are few new insecticides being developed and marketed for vector control. Herein, we report on the toxicity of six novel insecticides to both adult and larval Aedes aegypti (L). and the toxicity of three novel insect growth regulators (IGRs) to larvae. Four insecticides were highly or moderately toxic to larvae with LC50 values of 16 (chlorfenapyr), 70 (hydramethylnon), 79 (indoxacarb), and 84 ng/ml (imidacloprid). Diafenthiuron and chlorfenapyr were moderately toxic to adult mosquitoes with LC50 values of 13 and 92 ng/cm2, respectively. Imidacloprid was strongly synergized by piperonyl butoxide (PBO) in Ae. aegypti adults, suggesting that neonicotinoids are intrinsically very toxic to adult mosquitoes (in the absence of detoxification). The effect of PBO on the toxicity in adults and larvae was considerably different, both in terms of the insecticides that were synergized (or antagonized for chlorfenapyr versus adults) and in terms of the degree of synergism. This result implies that the cytochrome P450s involved in metabolism of these insecticides are different between adults and larvae. Pyriproxyfen was confirmed as a potent IGR (EC50 of 0.0017 ng/ml) for mosquitoes, although tebufenozide lacked activity. The potential for use of these materials in mosquito control is discussed.

INSECTICIDE RESISTANCE IN CULEX PIPIENS FROM NEW YORK

ABSTRACT Insecticides are the primary means to control Culex pipiens, an enzootic vector of West Nile virus, in the USA. To better understand how the evolution of resistance might impact control of this insect, we investigated the levels of resistance in populations collected from 2 metropolitan areas (Albany and Syracuse, NY) to 4 larvicides (methoprene, phenothrin, Bacillus sphaericus [Bs], and Bacillus thuringiensis israelensis [Bti]) and 1 adulticide (phenothrin) registered for mosquito control in New York State. High levels of resistance were found only to Bti, and only at 1 site (Syracuse). Resistance levels to the other insecticides were less than 10-fold. Given the large difference in Bti resistance between Syracuse and Albany, it appears these populations of Cx. pipiens do not rapidly mix, leading to localization of resistant populations.

Difference in the picrotoxinin receptor between the cyclodiene-resistant and susceptible strains of the german cockroach

Abstract As a result of toxicity tests, it was established that all cyclodiene-resistant strains of the German cockroach are also resistant to picrotoxinin, a plant-origin neurotoxicant. Two of the cockroach strains which exhibit a distinct cross-resistance pattern to picrotoxinin (i.e., LPP and FRP) are the ones that have been purified genetically by backcrossing against the susceptible (CSMA) strain. This cross-resistance pattern appears to be specific to picrotoxinin and does not extend to other neuroexcitants such as bicuculline, beta-bungarotoxin, and DDT. The nervous system of the resistant cockroach was found to be less sensitive to picrotoxinin. Furthermore, it was determined that nerve components from the resistant cockroaches have significantly lower binding capacity to [ 3 H]α-dihydropicrotoxinin. The most likely explanation for the above phenomenon is that these cockroaches have developed the cyclodiene resistance by altering the nerve receptor for picrotoxinin.