Spectrum of cross-resistance to insecticides in field samples of the primary sheep blowfly, Lucilia cuprina

Hughes P. B. (1981) Spectrum of cross-resistance to insecticides in field samples of the primary sheep blowfly, Lucilia cuprina. Internationaljournal for Parasitology11:475–479. Resistance to the organophosphorous (OP) insecticide, diazinon, is due to a major gene on chromosome 4. To examine the spectrum of cross-resistance in field populations of Lucilia cuprina, larvae from 41 field samples were exposed to the susceptible discriminating doses of five insecticides. A comparison of the mortalities produced by diazinon to those caused by the other OPs tested (fenthion-ethyl, chlorfenvinphos and coumaphos) indicates that the diazinon resistance gene may confer cross-resistance to other OPs used for blowfly control. However, two samples were found to be resistant to diazinon but susceptible to coumaphos. The OP resistance gene is present at a high frequency in field populations, with a mean frequency of 0–82. Mortalities produced by the carbamate, butacarb, did not relate to those due to diazinon. It seems that a separate mechanism for carbamate resistance is also present in the field. No indication of resistance was found to the insect growth regulator Vetrazin^® in any sample tested.     

Inheritance of resistance to chlorpyrifos in the Mt Alford strain and to diazinon in the Gracemere strain of the cattle tick (Boophilus microplus)

Reciprocal crossing of the Mt Alford (A) strain of the cattle tick B. microplus with a susceptible (S) strain and phenotype analysis of F1, testcross and F2 progeny showed that high chlorpyrifos resistance in strain A was due to two genes that were complementary and jointly exhibited incomplete dominance. Diazinon resistance in the Gracemere (G) strain appeared to be similarly inherited. The 'average' degree of dominance ('average dominance', Dav) of high chlorpyrifos resistance over susceptibility, exhibited by F1 hybrids from A X S reciprocal crossings, was +0.54 on a -1 to +1 scale and was not significantly different from the parametric value of +0.5 (semi-dominance). The corresponding Dav values revealed by G X S crossings were +0.42 for diazinon resistance (significantly less than +0.5) and -0.031 for chlorpyrifos resistance (not significantly different from zero and therefore exhibiting zero dominance/recessivity). Resistance factors for chlorpyrifos in strains A and G for homozygotes were 74 and 35, respectively, and for F1 hybrids were 25-29 and 5-7, respectively. The resistance factors for diazinon in strain G for homozygotes and F1 hybrids were 174 and 37-41, respectively.     

Ovicidal activity of topically applied acaricides against eggs of the southern cattle tick (Acari: Ixodidae)

Ovicidal activity of coumaphos, diazinon, dioxathion, chlorpyrifos, and crotoxyphos applied topically at 0.125, 0.25, and 0.5% (AI) to 1-d-old, 10-d-old, and 20-d-old eggs of southern cattle tick, Boophilus microplus (Canestrini), was determined. All acaricides produced some disruption of hatching of eggs in all age classes. Coumaphos, diazinon, dioxathion, and chlorpyrifos were most effective in reducing egg hatch of 1-d-old eggs but had little effect against 10- or 20-d-old eggs. Although crotoxyphos was comparable with other acaricides in reducing hatch in 1-d-old eggs, it caused significantly greater mortality (greater than or equal to 94.6%) in 20-d-old eggs than other compounds tested. Crotoxyphos applied at 0.5% (AI) is the only acaricide tested that provided sufficient ovicidal activity in all three egg age classes (greater than or equal to 85% reduction of hatch).     

Effect of selection pressure on the cholinesterase of Boophilus microplus (Acari: Ixodidae) resistant to coumaphos

The effect of selection pressure on the cholinesterase (AChE) activity of two strains of Boophilus microplus (Canestrini) resistant to coumaphos was monitored. Total AChE and protein was determined from three generations of resistant ticks and a susceptible strain. The effect of an AChE inhibitor, coroxon (the oxygen analog of coumaphos), was also determined. The resistance of the susceptible strain (Escondido) to coumaphos remained relatively unchanged throughout the study. The Tuxpan strain lost some of its resistance to coumaphos as the generations proceeded (AChE increased instead of decreased). The Tuxtla strain became more resistant to coumaphos as the generations proceeded (AChE increased).     

Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae)

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).     

Cyromazine resistance in the house fly (Diptera: Muscidae): genetics and cross-resistance to diflubenzuron

Larvae of a house fly, Musca domestica L., strain collected in a chicken house near Pittsburg, Tex, after a control failure with the poultry feedthrough insecticide cyromazine showed 6.5-fold resistance to cyromazine and 10-fold resistance to diflubenzuron. Adults of the strain showed high levels of resistance to carbaryl, DDT, and diazinon; moderate resistance to cypermethrin and permethrin; and low resistance to dieldrin. In contrast, no resistance to cyromazine was observed in eight laboratory house fly strains with resistance to four groups of conventional insecticides. When the genetics of cyromazine resistance was investigated in crosses to susceptible strains with visible mutant markers, results indicated cyromazine resistance was incompletely dominant over susceptibility and the resistance gene was on chromosome V. The same or a closely linked gene conferred resistance to diflubenzuron. A strain containing only chromosome V from the original resistant strain was resistant to cyromazine and diflubenzuron, but not to other insecticides except for low level resistance to DDT and carbaryl. Resistance to the latter insecticides appeared to be due to a linked, but distinct, gene. Therefore, resistance to cyromazine and probably diflubenzuron appears to be genetically distinct from other types of insecticide resistance.     

Identification of a point mutation in an esterase gene in different populations of the southern cattle tick, Boophilus microplus

Two esterase cDNA sequences were obtained from susceptible and organophosphorus resistant strains of Boophilus microplus. Both sequences have a high degree of homology to carboxylesterase B. One gene has identical sequences in both strains and the other showed two point mutations. One mutation produces an amino acid substitution when the amino acid sequence is deduced, this mutation was detected in six different populations susceptible and resistant to insecticides, but a pyrethroid resistant strain was the only one that showed only the mutant allele. Identification of this mutation and the strong signal detected in southern blot with this strain, suggest that esterases are contributing to detoxification of pyrethroid compounds, as a resistant mechanism in Mexican strains of the southern cattle tick.     

Diagnosis of amitraz resistance in Brazilian populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) with larval immersion test

Among the ectoparasites of cattle, Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae) remains a major cause of economic losses to livestock. The chemical control with acaricides is still the most efficient method available to control ticks. The aims of this study were to diagnose resistance to amitraz in 16 tick populations from the States of São Paulo (14) and Paraná (2), using the larval immersion technique (LIT), and evaluate the effect of synergists [piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP)] on amitraz resistant and susceptible strains of cattle tick. Most of the evaluated populations (68.7 %) showed to be resistant to amitraz, with resistance ratios ranging from 2.14 to 132. The results suggest that the test procedure by LIT is sensitive and adequate for detection and monitoring of amitraz resistance in cattle tick. No synergistic effect was observed for the synergists PBO, DEM and TPP, on the amitraz resistant (Poa) strain of cattle tick, indicating that increased detoxification metabolism was not involved in this resistance.     

Factors affecting the toxicity of diazinon to Musca domestica L

Processes affecting the toxicity of diazinon to a susceptible and a resistant strain of houseflies were examined. More evidence was obtained to show that slower penetration of diazinon through the integument of resistant flies is a cause of resistance. Small amounts of two decomposition products were found in both strains. The decomposition mechanisms, in these strains were differently distributed and, although detoxication of diazinon in the two strains is quantitatively similar and small, it may contribute to resistance. Traces of diazoxon were detected when diazinon was incubated with tissue extracts of either strain. Tissue extracts of resistant, but not of susceptible, flies decomposed significant amounts of diazinon in 1 hr. and the ability to decompose diazoxon seems to be an important cause of resistance. Tissues of both strains sorbed diazinon from aqueous solution similarly; the quantities sorbed were large and suggest that sorption may increase the amount of poison needed inside the insects to kill, by between five and forty times.     

Acaricide resistance status in Indian isolates of Hyalomma anatolicum

The multi host tick, Hyalomma anatolicum, is the commonest Hyalomma species in India and cattle serves as the main host of this species. A study to evaluate the acaricide resistance of H. anatolicum to deltamethrin, cypermethrin and diazinon was conducted in 20 areas located in three agro climatic regions known to have abundance of the species. Results obtained by the “larval packet test” (LPT) showed a low grade resistance (level-I, RF?<5) in the tick species to both deltamethrin and cypermethrin in 10 areas and higher grade resistance (level-II, RF?<25) to deltamethrin in one area, where intensive use of synthetic pyrethroids are practiced for tick control. Low grade resistance to diazinon (level I) was recorded in six areas where organophosphates compounds are extensively used for agricultural practices allowing increased exposure of the moulting instars of the ticks to these chemicals. Biochemical analysis of the samples suggested involvement of esterase and alterations of acetylcholinesterase in the resistance mechanisms.     

Aspects of insecticide resistance in the melon and cotton aphid, Aphis gossypii (Hemiptera: Aphididae)

Strains of the aphid Aphis gossypii, taken from cucumber in UK glasshouses were tested for resistance to pirimicarb, diazinon and heptenophos. Compared to a susceptible strain, resistance factors to diazinon of up to 659 were found in populations collected between 1987 and 1989. Compared to the same susceptible strain, diazinon-resistant populations also showed a reduced response to heptenophos. All field populations tested against pirimicarb were resistant. Host plant preference tests showed that all strains (including stock cultures) tested could readily breed on cotton. However strains originally collected from chrysanthemum did not transfer to cucumber and likewise strains from cucumber would not colonise chrysanthemum. Polyacrylamide gel electrophoresis of four of the aphid strains showed differences in migration rates and intensity of staining of esterase bands. Intensity of staining was correlated with the degree of diazinon resistance.     

Identification of a point mutation in the para-type sodium channel gene from a pyrethroid-resistant cattle tick.

To investigate the molecular mechanism of resistance to pyrethroids in the southern cattle tick, Boophilus microplus, we have obtained and sequenced a partial para-homologous sodium channel cDNA from susceptible and pyrethroid-resistant tick strains. A point mutation that results in an amino acid change from Phe to Ile was identified in the highly conserved domain IIIS6 of the homologous sodium channel from ticks that are highly resistant to pyrethroid acaricides. This mutation is at a location different from those reported in the same gene in pyrethroid-resistant insects.     

Monitoring for resistance to organophosphorus and pyrethroid insecticides in Varroa mite populations

The occurrence of resistance in Varroa mite populations is a serious threat to the beekeeping industry and to crops that rely on the honey bee for pollination. Integrated pest management strategies for control of this pest include the judicious use of insecticides. To monitor field populations of Varroa mite for insecticide resistance, a glass vial bioassay procedure was developed to use in the development of a resistance management strategy. Diagnostic concentrations needed to separate susceptible genotypes from resistant individuals were determined for cypermethrin (0.1 microg per vial), fluvalinate (5.0 microg per vial), malathion (0.01 microg per vial), coumaphos (10.0 microg per vial), diazinon (5.0 microg per vial), methomyl (0.5 microg per vial), propoxur (0.1 microg per vial), and endosulfan (2.5 microg per vial). Resistance to organophosphorus insecticides (malathion, coumaphos) and pyrethroids (cypermetrhrin, fluvalinate) was widespread in both La Media Ranch, TX, and Wewahitchka, FL, from 2007 to 2009. There was no resistance to endosulfan, diazinon, methomyl, and propoxur in field populations of Varroa mite in the two locations where resistance was monitored. The seasonal patterns of resistance in Wewahitchka were different from those of La Media Ranch. In the former location, the frequency of resistance to all insecticides tested decreased significantly from 2007 to 2009, whereas it increased in the latter location. Resistance levels were unstable, suggesting that resistance could be successfully managed. The results validate use of the glass vial bioassay to monitor for resistance in Varroa mite and provide the basis for the development of a resistance management strategy designed to extend the efficacy of all classes of insecticides used for control of Varroa mite.     

Molecular mechanism of synthetic pyrethroid and organophosphate resistance in field isolates of <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick collected from a northern state of India

The frequently used chemical control method to manage Rhipicephalus microplus is limited by the emergence of resistance populations. Understanding of resistance mechanisms is essential to develop strategy for sustainable management. The present study was focused on working out the molecular mechanisms of resistance against synthetic pyrethroids (SPs) and organophosphates (OPs) in field isolates of R. microplus collected from six districts of Uttar Pradesh, India. Adult immersion test with discriminating concentrations (AIT-DC) was used to determine resistance status of isolates to SPs (deltamethrin, cypermethrin) and OPs (diazinon, coumaphos). All the six isolates were found resistant to SPs with resistance factor (RF) of 2.9–58.6 and to one of the OP compounds, diazinon having RF of 3.5–13.7 but susceptible to coumaphos (RF?<?1.4). Three R. microplus genes, viz. para-sodium channel domain II S4-5 linker, carboxylesterase (372 bp) and acetylcholinesterase 2 (1692 bp) were sequenced and compared with respective sequences of reference susceptible IVRI-I, reference OP resistant population (IVRI-III), IVRI-IV and multi-acaricide resistant population (IVRI-V) of R. microplus. A C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to L64I amino acid substitution was detected in all six isolates. The G1120A mutation in the carboxylesterase gene could not be detected in any isolate. Five nucleotide substitutions viz., G138A, G889A, T1090A, C1234T and G1403A were identified in the acetylcholinesterase 2 gene leading to four amino acid substitutions. The findings of the study corroborate the role of mutation in sodium channel and acetylcholinesterase 2 genes in SP and OP resistance in this part of India.     

Resistance in cattle againstRhipicephalus appendiculatus with an assessment of cross-resistance toR. pulchellus (Acari: Ixodidae)

A comparison of the yield of engorged nymphs ofRhipicephalus appendiculatus andR. pulchellus was carried out between cattle highly resistant toR. appendiculatus but susceptible toR. pulchellus, and cattle and rabbits susceptible to both species.Resistant cattle yielded fewer and smallerR. appendiculatus nymphs than either susceptible cattle or rabbits. More nymphs of both tick species dropped from susceptible cattle but no differences were detected in the mean weight of engorgedR. pulchellus nymphs fed onR. appendiculatus-resistant and susceptible cattle, although resistant animals yielded more nymphs ofR. pulchellus than ofR. appendiculatus.An acute inflammatory reaction, accompanied by intense pruritus and grooming, was observed in the ears of the resistant cattle infested withR. appendiculatus, but no reaction was detected in the opposite ears whereR. pulchellus was feeding.The low cross-resistance between these two closely related tick species suggests that different antigens may be involved in the development of resistance in cattle to different tick species.     

Effects of acute sublethal exposure to coumaphos or diazinon on acquisition and discrimination of odor stimuli in the honey bee (Hymenoptera: Apidae)

Two organophosphate compounds, coumaphos and diazinon, were examined for effects of sublethal exposure on odor learning and generalization in honey bees, Apis mellifera L. Using proboscis extension response training as a measure of odor learning and discrimination, a series of two experiments tested whether these compounds would inhibit bees from learning a new odor or discriminating between different odors. Bees were exposed to coumaphos or diazinon in acetone applied to the thorax, or to coumaphos or diazinon in hexane injected intracranially. At no dose tested or exposure method used was coumaphos shown to inhibit acquisition of a novel odor stimulus, although it was shown to slightly reduce discriminatory ability when given by intracranial injection. Diazinon had effects on odor learning at several small doses, and a small injected dose was shown to significantly inhibit learning of an odor stimulus paired with a sucrose reward. When bee head acetylcholineasterase activity was measured after dermal applications of both pesticides, only the higher doses of diazinon showed reduced activity, indicating that externally-applied coumaphos shows no significant effect on bee brain acetylcholinesterase activity. These data suggest that acute application of coumaphos has only slight nonlethal effects upon the behavior of honey bees and should have little effect upon bee tasks that involve odor learning.     

Tick-susceptible Bos taurus cattle display an increased cellular response at the site of larval Rhipicephalus (Boophilus) microplus attachment, compared with tick-resistant Bos indicus cattle

Cattle demonstrate divergent and heritable phenotypes of resistance and susceptibility to infestation with the cattle tick Rhipicephalus (Boophilus) microplus. Bos indicus cattle are generally more resistant to tick infestation than Bos taurus breeds although large variations in resistance can occur within subspecies and within breed. Increased tick resistance has been previously associated with an intense hypersensitivity response in B. taurus breeds; however, the mechanism by which highly resistant B. indicus cattle acquire and sustain high levels of tick resistance remains to be elucidated. Using the commercially available Affymetrix microarray gene expression platform, together with histological examination of the larval attachment site, this study aimed to describe those processes responsible for high levels of tick resistance in Brahman (B. indicus) cattle that differ from those in low-resistance Holstein-Friesian (B. taurus) cattle. We found that genes involved in inflammatory processes and immune responsiveness to infestation by ticks, although up-regulated in tick-infested Holstein-Friesian cattle, were not up-regulated in Brahman cattle. In contrast, genes encoding constituents of the extracellular matrix were up-regulated in Brahmans. Furthermore, the susceptible Holstein-Friesian animals displayed a much greater cellular inflammatory response at the site of larval R. microplus attachment compared with the tick-resistant Brahman cattle.     

Enzyme-based strategy for toxic waste treatment and waste minimization

The increasing amounts of pesticides used throughout the world, as well as the increasingly stringent governmental regulations concerning waste disposal, mandates improved techniques of waste disposal and minimization. In this article, parathion hydrolase, an enzyme with proven effectiveness at hydrolyzing organophosphates, was used to treat a cattle dipping liquid containing the pesticide, coumaphos, which is used to kill a disease-causing tick. Waste is generated from this process when a toxic dechlorination product of coumaphos, potasan, accumulates to concentrations hazardous to the cattle. This pesticide system was used as a model to demonstrate how enzyme technology can be applied to waste treatment and minimization. Kinetic experiments showed that the hydrolysis of the two organophosphate substrates can be modeled as first-order reactions with identical rate constants. It was further shown that the enzyme is capable of hydrolyzing only dissolved substrates. Because of the eightfold greater solubility of potasan than coumaphos (16.9 vs. 2.2 mumol/L), it was possible to utilize the enzyme to hydrolyze potasan selectively. Thus, by limiting the amount of enzyme, it is possible to remove potasan selectively to extend the lifetime of the cattle dipping liquid, thereby reducing the amount of waste generated. Based upon experimental results, a mathematical model describing the system was developed and verified. The mathematical model was then used to simulate the ability of the enzyme to hydrolyze the total amount of organophosphates, and to degrade selectively all of the toxic potasan without a significant loss of coumaphos.     

Molecular and Biochemical Diagnosis of Esterase-Mediated Pyrethroid Resistance in a Mexican Strain of Boophilus microplus (Acari: Ixodidae)

We examined pyrethroid resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to determine the mechanisms conferring resistance. Permethrin hydrolysis assays and esterase activity gels indicated enhanced esterase-mediated metabolic detoxification in the Cz strain, while one other pyrethroid resistant strain, SF, and two pyrethroid susceptible strains had lower levels of permethrin hydrolysis. Results from assays using a PCR-based test to detect a pyrethroid target site resistance-associated mutation in the tick sodium channel gene found only low levels of mutations in the Cz strain, while the SF strain had a high level of the mutated sodium channel alleles. A specific esterase, designated CzEst9, believed to be responsible for the esterase-mediated pyrethroid resistance in the Cz strain was purified, and the gene encoding CzEst9 cloned. This revised version was published online in July 2006 with corrections to the Cover Date.