George P. Georghiou

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Management of Resistance in Arthropods

Since the earliest days of their awareness of resistance, entomologists have been concerned with understanding the factors responsible for its development and with divising measures for its control. It is remarkable that in reporting the first case of resistance — in the San Jose scale toward lime sulfur — Melander (1914) recognized the role of incomplete coverage and genetic recessiveness and speculated that should the scale become resistant also to oil sprays “we might have to introduce a weak strain to cross with the immune and thus return to the normal susceptible population.” Melander and other early pioneers in studies of resistance (Quayle 1922, Woglum 1925) may have been ahead of their time, however, for in the subsequent 30 years or so, resistance evolved slowly, affecting only 12 species of arthropods (review by Babers 1949). Interest in resistance intensified with the introduction of DDT and with the rapid development in cases of resistance to organochlorine, organophos-phate, carbamate and most recently to pyrethroid insecticides. The phenomenon now involves at least 428 species of arthropods and every class of commonly available compound (Georghiou and Mellon, this volume).

Pesticide Resistance in Time and Space

Within the evolutionarily insignificant period of just 65 years, beginning when the first case of resistance to a pesticide was reported (Melander, 1914), the phenomenon of resistance has proliferated exponentially so as to constitute today an indispensable consideration in nearly every pest control program.

Amplification of various esterase B's responsible for organophosphate resistance in Culex mosquitoes.

Increased detoxification by esterases is a common mechanism of resistance to organophosphate (OP) insecticides in insects. Utilizing a partial cDNA of this gene, we probed genomic fragments generated by EcoR1 restriction enzyme in various laboratory and natural populations of Culex that are OP resistant

Insecticide resistance and prospects for its management.

After a decade of relative neglect, resistance to insecticides1 is again beginning to receive the attention it deserves as a challenging problem that requires new solutions.

Genetic and biochemical studies of the highly active esterases A′ and B associated with organophosphate resistance in mosquitoes of the Culex pipiens complex

The highly active esterases A′ and B that cannot be dissociated from OP resistance in Culex pipiens from France and California are shown to have equivalent Km values (2.1×10−6m/min/mosquito) but different turnover rates (Vm=2.13 and 0.57×10−6m/min/mosquito, respectively) and pH for maximum activity. Both enzymes have broad substrate specificities and at least one, esterase A′, can hydrolyze OP insecticides. In addition, esterases A′ and B are coded by two closely linked genes, Est-3 and Est-2, respectively (0.67 unit of crossing over), located on the same autosome as pl, a locus attributed to linkage group III. The estimated distance between Est-2 and pl was 9.4 units.

Mechanisms of resistance to diflubenzuron in the house fly, Musca domestica (L.)

Abstract The mechanisms of resistance to the chitin synthesis inhibitor diflubenzuron were investigated in a diflubenzuron-selected strain of the house fly ( Musca domestica L.) with > 1000 × resistance, and in an OMS-12-selected strain [ O -ethyl O -(2,4-dichlorophenyl)phosphoramidothioate] with 380 × resistance to diflubenzuron. In agreement with the accepted mode of action of diflubenzuron, chitin synthesis was reduced less in larvae of the resistant (R) than of a susceptible (S) strain. Cuticular penetration of diflubenzuron into larvae of the R strains was about half that of the S. Both piperonyl butoxide and sesamex synergized diflubenzuron markedly in the R strains, indicating that mixed-function oxidase enzymes play a major role in resistance. Limited synergism by DEF ( S,S,S -tributyl phosphorotrithioate) and diethylmaleate indicated that esterases and glutathione-dependent transferases play a relatively small role in resistance. Larvae of the S and R strains exhibited a similar pattern of in vivo cleavage of 3 H- and 14 C-labeled diflubenzuron at N 1 C 2 and N 1 C 1 bonds. However, there were marked differences in the amounts of major metabolites produced: R larvae metabolized diflubenzuron at considerably higher rates, resulting in 18-fold lower accumulation of unmetabolized diflubenzuron by comparison with S larvae. Polar metabolites were excreted at a 2-fold higher rate by R larvae. The high levels of resistance to diflubenzuron in R-Diflubenzuron and R-OMS-12 larvae are due to the combined effect of reduced cuticular penetration, increased metabolism, and rapid excretion of the chemical.

Laboratory selection for resistance to Bacillus sphaericus in Culex quinquefasciatus (Diptera: Culicidae) from California, USA.

Abstract A previously untreated field population ofCulex quinquefasciatusSay, collected near Bakersfield, CA, was subjected to intensive laboratory selection with the bacterial insecticideBacillus sphaericusNeide (strain 2362) at a level producing 95% mortality. Resistance rapidly appeared and resistance levels increased such that fourth instars of generation 12 were able to survive a concentration ofB. sphaericusthat was 7,000 times higher than the median lethal concentration (LC50) of the susceptible reference colony. Similar resistance levels were detected in first instars. Cross-resistance in the selected colony was detected towardB. sphaericusstrains 1593 and 2297, but little or no cross-resistance was observed towardB. sphaericusstrains IAB59 or ISPC5 (=WHO 2173). Cross-resistance also was not detected toward the bacterial insecticideBacillus thuringiensissubsp.israelensis,toward a recombinant strain expressing bothB. thuringiensissubsp.israelensisandB. sphaericus(strain 1593) toxins, toward individual or multiple toxins fromB. thuringiensissubsp.israelensis,or toward conventional synthetic insecticides. Genetic analysis revealed thatB. sphaericusresistance was inherited as a recessive trait and controlled by a single major locus. These data are discussed in relation to cases of field resistance toward this biopesticide in theCx. pipiens(L.) complex.

Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides

Abstract Acetylcholinesterase from fourth instar Anopheles albimanus larvae was studied in vitro . The acetylcholinesterase from both the resistant and susceptible strains behaved as a single enzyme “type,” with straight pseudo first-order insecticide inhibition lines which intersected the Y axis at 100%. The enzyme from resistant larvae was more slowly inhibited than the susceptible enzyme; bimolecular rate constants ( k i ) differed by approximately 1.2- to 6-fold for a range of organophosphorous compounds and 17- to 1570-fold for the carbamates. There was a good correlation between the levels of resistance and the acetylcholinesterase inhibition rates.

Decreased nerve sensitivity and decreased cuticular penetration as mechanisms of resistance to pyrethroids in a (1R)-trans-permethrin-selected strain of the house fly

Abstract A fenthion-resistant strain of the house fly ( Musca domestica L.) was selected with bioresmethrin resulting in ca. 90-fold resistance to the selecting agent. This strain was subsequently selected with (1 R )- trans -permethrin producing ca. 140-fold resistance to this latter insecticide. The permethrin-resistant (147-R) strain was highly cross-resistant to several other pyrethroids and demonstrated resistance to knockdown by these insecticides as well as by DDT. The sensitivity of the central nervous system to four pyrethroids was investigated. The 147-R strain was 2.6-fold less sensitive to (1 R )- trans -ethanoresmethrin than the susceptible (NAIDM-S) strain, and >43-fold and >67-fold less sensitive to (1 R,S )- cis, trans -tetramethrin and (1 R )- trans -permethrin, respectively. It also displayed decreased penetration of (1 R,S )- trans -[ 14 C]permethrin when compared to the NAIDM-S strain. Lower nerve sensitivity and decreased cuticular penetration are potential mechanisms of resistance to pyrethroids in house flies in the United States.