Enhancement of dye-sensitized phototoxicity to house fly larvae in vivo by dietary ascorbate,diazabicyclooctane, and other additives

Several reagents reported to be singlet oxygen quenchers and/or radical scavengers and to be protectants against photooxidative damage to isolated systems in vitro were examined, in vivo, for protective effect on the larvae of the house fly (Musca domestica L.; Diptera:Muscidae). A standardized erythrosine-sensitized phototoxic test procedure was used. β-Carotene appeared to show some protective effect. Other dietary additives exhibited no measurable degree of protection to the larvae in vitro; on the contrary, mortality increased in the presence of butylated hydroxytoluene, ascorbate, and diazabicyclooctane.     

Neural and behavioral correlates of pyrethroid and DDT-type poisoning in the house fly, Musca domestica L

Intact house flies were observed during poisoning caused by several pyrethroid and DDT-type insecticides. The two insecticide classes could be generally distinguished from each other based on differences in symptoms and several physiological correlates. Both insecticide types caused motor unit repetitive backfiring, but the temporal development and stability of repetitiveness were distinctly different between the two classes. Repetitive backfiring always disappeared at low temperatures, but DDT-type backfiring disappeared at lower temperatures than the pyrethroids. trans-Tetramethrin caused a threshold increase in flight motor nerve endings which did not occur in DDT or trans-Barthrin poisoning. Pyrethroids caused “uncoupling” of the flight motor pattern, while DDT-types did not. trans-Barthrin, a methylenedioxyphenyl pyrethroid, was unique in causing both symptoms and physiological aberrations which more closely resembled those of the DDT-types than the pyrethroids.     

Indoxacarb resistance in the house fly, Musca domestica

Indoxacarb (DPX-MP062) is a recently introduced oxadiazine insecticide with activity against a wide range of pests, including house flies. It is metabolically decarbomethoxylated to DCJW. Selection of field collected house flies with indoxacarb produced a New York indoxacarb-resistant (NYINDR) strain with >118-fold resistance after three generations. Resistance in NYINDR could be partially overcome with the P450 inhibitor piperonyl butoxide (PBO), but the synergists diethyl maleate and S,S,S-tributyl phosphorothioate did not alter expression of the resistance, suggesting P450 monooxygenases, but not esterases or glutathione S-transferases are involved in the indoxacarb resistance. Conversely, the NYINDR strain showed only 3.2-fold resistance to DCJW, and this resistance could be suppressed with PBO. Only limited levels of cross-resistance were detected to pyrethroid, organophosphate, carbamate or chlorinated hydrocarbon insecticides in NYINDR. Indoxacarb resistance in the NYINDR strain was inherited primarily as a completely recessive trait. Analysis of the phenotypes vs. mortality data revealed that the major factor for indoxacarb resistance is located on autosome 4 with a minor factor on autosome 3. It appears these genes have not previously been associated with insecticide resistance.     

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

Toxicokinetics of carbofuran in house fly, Musca domestica L

The kinetics of accumulation and elimination of lethal doses of [¹⁴C]carbofuran in the hemolymph of the house fly suggest a one-compartment open model. Carbofuran in the hemolymph appeared to be in equilibrium with that in the tissues very soon after treatment.Following topical application of carbofuran, the rate of onset of symptoms of poisoning was correlated with the amount of carbofuran in the hemolymph, and the onset of convulsions only occurred after the concentration of carbofuran in the hemolymph reached μM levels. This value correlated well with neurobioassays of known concentrations of carbofuran perfused in saline onto the isolated thoracic ganglion.Following topical doses, carbofuran concentration in the hemolymph reached a peak within an hour and then gradually declined. At an LD₆₀ dose, the initial decline in carbofuran concentration in the hemolymph over time was significantly slower than the decline after an LD₁₀ dose, suggesting saturation kinetics.Hemolymph was collected from house flies for up to 3 hr following topical application of toxic amounts of carbofuran. Thereafter, hemolymph volume decreased and blood samples could not be collected. Curiously, hemolymph samples could be collected for 5 hr from house flies that were injected with toxic doses of carbofuran.     

In vivo neurotoxic effects of dieldrin on giant nerve fibers and escape reflex function in the earthworm, Eisenia foetida

Neurotoxicological effects of dieldrin were assessed in adult earthworms, Eisenia foetida, using noninvasive electrophysiological recordings of escape reflex activity. After 48 hr body surface exposure to aqueous suspensions of dieldrin, dose-dependent reductions in medial and lateral giant nerve fiber conduction velocities were seen within a concentration range of 1.4 to 288 ppm. At a high concentration (288 ppm) the decreases in velocity occurred in as little as 90 min, but at low concentrations decreases in velocity occurred much more gradually. Other physiological and behavioral effects of dieldrin treatment included (i) after-discharges of giant fibers following brief mechanical stimulation, (ii) spontaneous bursts of giant fiber spikes, (iii) reductions in the amplitude of giant fiber-mediated muscle potentials, (iv) tonic spasms involving end-to-end shortening and body stiffening, (v) ataxia, and (vi) reduction in body weight. Thus, the in vivo effects of dieldrin in earthworms are characterized by a diverse and complex set of physiological and behavioral symptoms. Possible mechanisms underlying these effects are discussed in relation to the generally accepted mode of dieldrin action on synaptic transmission.     

In vitro metabolism of insecticides during midgut penetration

A study of the metabolism of ¹⁴C-labeled dieldrin, DDT, malathion, and carbaryl during penetration of the isolated midgut of two insects (Blaberus discoidalis and Manduca sexta) and a section of the intestine of a mammal (Mus musculus) is reported. There was appreciable metabolism of malathion during penetration, including differences in the activation reaction to malaoxon, between insects and mammals. Metabolism was relatively slow during penetration of carbaryl and the chlorinated hydrocarbon insecticides, and little difference in metabolic patterns was noted among the three species. The penetration studies were supported by experiments in which insecticides were incubated with intact and homogenized midgut preparations.     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

In vitro metabolism of EPN and EPNO in susceptible and resistant strains of house flies

EPN is twice as toxic as EPNO to house flies from both the Diazinon-resistant strain and the susceptible strain. EPN and EPNO are also eight times more toxic to the susceptible than the resistant strain. This is due to the ability of the resistant strain to metabolize these compounds to a greater extent. Metabolism by the glutathione S-transferases present in the 100,000g supernatant is more extensive than that by the NADPH-dependent microsomal mixed-function oxidases. The glutathione S-transferases are the major route of metabolism for EPN and appear to be the principal mechanism conferring resistance. EPN was metabolized by the microsomal fraction via oxidative desulfuration to the oxygen analog, EPNO, and by oxidative dearylation to p-nitrophenol. EPNO was metabolized by the same system to p-nitrophenol and desethyl EPNO as well as to an unknown metabolite. The soluble fraction metabolized EPN to p-nitrophenol, S-(p-nitrophenyl)glutathione, O-ethyl phenylphosphonothioic acid, and S-(O-ethyl phenylphosphonothionyl)glutathione. The identification of the latter conjugate demonstrates a new type of metabolite of organophosphorus compounds. EPNO was metabolized by the soluble fraction to p-nitrophenol and S-(p-nitrophenyl)glutathione.     

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.     

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

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 ³H- and ¹⁴C-labeled diflubenzuron at N₁C₂ and N₁C₁ 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.     

Characterization of multiple glutathione transferases from the house fly, Musca domestica (L)

Glutathione transferases have been purified to a high degree of homogeneity from three strains of house fly by a procedure involving affinity chromatography on glutathione-sulfobromophthalein conjugate immobilized on Sepharose 4B, followed by preparative isoelectrofocusing. The affinity chromatography yielded purifications of between about 10- and 100-fold, depending on the strain and the substrate with which activity was measured. Each strain was shown to possess several proteins with glutathione S-transferase activity which fell into two clearly defined groups. The first group, of relatively low isoelectric point, showed activity with CDNB but little with DCNB, p-nitrobenzylchloride, or 1,2-epoxy-3-(p-nitrophenoxy)propane, whereas the second group, of higher isoelectric points, showed substantial activity with all substrates tested. Studies on the subunit structure of these enzymes demonstrated the existence of three different sized subunits of M_r 20,000, 22,000, and 23,500. From the experimental evidence recorded here, the existence of at least three functionally different glutathione transferases is inferred.     

In vitro inhibition of Sclerotinia sclerotiorum by mixtures of azoxystrobin, SHAM, and thiram

The necrotrophic fungal phytopathogen Sclerotinia sclerotiorum (Lib.) de Bary has a broad host range and frequently causes destructive diseases. The extensive use of common fungicides to control these diseases has selected for resistance in populations of S. sclerotiorum. In this study, 105 isolates of S. sclerotiorum from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to azoxystrobin, and the average EC₅₀ value was 0.2932 μg/mL for mycelial growth. Of the mixtures of the fungicides thiram and azoxystrobin that were tested using an in vitro mycelial growth assay, the 1:4 ratio provided the greatest inhibition of S. sclerotiorum. When tested against nine isolates, the 1:4 mixture resulted in a mean synergy ratio of 2.31, indicating synergistic inhibition. Mycelial respiration was inhibited for about 2 h by azoxystrobin alone but for 48 h by the mixture of thiram and azoxystrobin. Salicylhydroxamic acid (SHAM, a known inhibitor of alternative respiration) also increased the inhibition of mycelial growth and respiration caused by azoxystrobin. These results suggest the need for further study of effects of combinations of azoxystrobin with thiram or SHAM in planta to evaluate their potential for management of diseases caused by S. sclerotiorum.     

Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica L

The mechanisms of resistance and cross resistance to the juvenoids methoprene and R-20458 in the house fly, Musca domestica, were examined. Radiolabeled methoprene was found to be metabolized faster in resistant and cross-resistant house fly larvae than in susceptible larvae, and methoprene and R-20458 penetrated more slowly into larvae of the resistant strain. In vivo and in vitro metabolism of methoprene was largely by oxidative pathways followed by conjugation in all strains examined, and little or no ester change of methoprene was noted in vitro. In vitro oxidative metabolism of methoprene, R-20458, juvenile hormone I, and several model substrates was higher in resistant and cross-resistant larvae than in susceptible larvae. Juvenoid functionalities susceptible to metabolic attack by resistant strains are indicated.     

Effects of temperature on fitness costs, insecticide susceptibility and heat shock protein in insecticide-resistant and -susceptible Plutella xylostella

Effects of high temperature on insecticide susceptibility and fitness were studied in the field population of Plutella xylostella (L.) (Lepidoptera: Plutellidae) (R DBM) and a susceptible field-insectarium population of P. xylostella (S DBM). R DBM displayed 18.3-fold resistance to methamidophos and 74.0-fold resistance to avermectin. The population growth tendency index (I) values were 41.8 (S DBM) and 27.7 (R DBM) at 25 °C, and 1.19 (S DBM) and 0.23 (R DBM) at 33.5 °C. The level of methamidophos resistance in the progenies of R DBM declined sharply when reared at high temperature for one generation. The increase of susceptibility to methamidophos appeared to pass from generation to generation. S DBM displayed higher up-regulation of Hsp70 expression at high temperature than R DBM. It was suggested that low fitness in R DBM caused by high temperature might be involved in the sharp decline of methamidophos resistance under high temperature conditions.     

The biochemistry of insecticide resistance in Anopheles sacharovi: Comparative studies with a range of insecticide susceptible and resistant Anopheles and Culex species

Fourth instar larvae, the progeny from wild-caught Anopheles sacharovi females, were subjected to a number of biochemical tests and the results were compared to those from similar tests on laboratory insecticide resistant and susceptible strains of anopheline and culicine mosquitoes. DDT resistance in An. sacharovi is associated with the ability to rapidly metabolise DDT to DDE. The organophosphorus and carbamate resistance was not associated with quantitative changes in esterases, multifunction oxidases, or glutathione S-transferase. The acetylcholinesterase was less sensitive to malaoxon and propoxur than laboratory susceptible An. albimanus, and plots of inhibition suggest that the population was polymorphic for more than one form of acetylcholinesterase. Metabolism studies on malathion and pirimiphos methyl did not indicate resistance due to increased metabolism. There was no evidence of penetration barriers contributing to resistance to either DDT or malathion, and there was no indication of any resistance to pirimiphos methyl in our tests.     

Frequencies and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and field populations of the house fly, Musca domestica L.

Resistance to organophosphate (OP) and/or carbamate insecticides can be due to mutations in the acetylcholinesterase gene (Ace). Genotypes of house fly, Musca domestica L., Ace were determined in twelve laboratory maintained strains (originally from North America, Europe and Asia) and two field collected populations from New York and Florida. There were 15 Ace alleles found and 11 of the alleles coded for a susceptible form of the enzyme (i.e., V260, A316, G342 and F407). Three of the four resistance alleles were previously described, while one is new. Phylogenetic analysis of the alleles suggests multiple origins of the F407Y mutation and multiple origins of the G342A mutation that confer OP resistance. Genotyping of field collected house flies from New York and Florida populations revealed the presence of only one resistance allele, Acev10 (containing the non-synonymous mutations for A342 and Y407). All other alleles detected from the field-collected flies coded for a susceptible AChE. Thus, we were able to categorize individual flies as having homozygous susceptible (Ace^S/Ace^S), homozygous insensitive (Ace^I/Ace^I or Acev10/Acev10) or heterozygous AChE. The frequencies of Ace^S and Ace^I were not different between the NY2002 and FL2002 populations. Both populations were out of Castle-Hardy-Weinberg equilibrium, having an excess of Ace^S/Ace^I individuals and very few Ace^S/Ace^S individuals. Comparison of Ace, Vssc and CYP6D1 genotypes indicates individual house flies commonly have resistance alleles at multiple loci. Comparison of genotype data with bioassays, as well as the use of genotype data in resistance studies is discussed.     

Structure-activity relationships of 1,2,3-benzothiadiazoles as synergists for carbaryl against the house fly (Musca domestica)

Fifty six 1,2,3-benzothiadiazoles and related compounds were evaluated as carbaryl synergists against the house fly (Musca domestica). Many of these were excellent synergists, the most active being those containing various combinations of halogen, alkyl, or alkoxy substituents in the 5- and/or 6-positions of the ring.Regression analysis on the data from 14 compounds for which substituents constants were available established that synergistic activity can be satisfactorily described by equations in terms of the hydrophobic bonding constant (π) and the homolytic free radical constant (σ ·).The results with compounds related to the 1,2,3-benzothiadiazoles suggest that synergistic activity is associated primarily with the diazosulfide moiety.     

DDT and pyrethroids: Receptor binding in relation to knockdown resistance (kdr) in the house fly

The nature of target site or knockdown resistance (kdr) to DDT and pyrethroids was studied by investigating specific binding of [14_C] DDT and [14_C] cis-permethrin to the previously established membrane receptors from the heads of susceptible (sbo) and resistant (kdr) strains of the house fly, Musca domestica L. In vivo studies showed the heads from sbo flies bound two to three times more DDT than those from kdr flies at all doses tested. Reduced binding was also observed in kdr flies in in vitro [14_C] DDT binding assays. Scatchard analysis indicated that kdr flies have the same affinity but fewer receptors per milligram protein in the CNS than sbo flies. Assays with [14_C] cis-permethrin also showed binding was much reduced in kdr flies in comparison with sbo flies. Based on these results, the nature of the target site insensitivity of kdr flies may relate to their having a reduced number of receptors for the insecticides.     

In vitro inhibition of α-amylase and protease by nematocides in Cicer arietinum L

The in vitro effects of four systemic nematocides, i.e., aldicarb, carbofuran, oxamyl, and phorate, on the α-amylase and protease activities in Cicer arietinum has been revealed. All four nematocides markedly inhibited the activities of both the enzymes, with a general tendency of increased inhibition with corresponding increase in the concentrations of the nematocides. There was complete inhibition of α-amylase activity by the highest concentration (500 μM) of aldicarb and carbofuran, while oxamyl at the same concentration showed the same effects on protease activity. The lowest concentration (10 μM) was almost ineffective.