J. Marshall Clark

Biochemical and Molecular Analysis of Deltamethrin Resistance in the Common Bed Bug (Hemiptera: Cimicidae)

Abstract This study establishes deltamethrin resistance in a common bed bug, Cimex lectularius L., population collected from New York City (NY-BB). The NY-BB population was 264-fold more resistant to 1% deltamethrin in contact bioassay compared with an insecticide-susceptible population collected in Florida (FL-BB). General esterase, glutathione S-transferase, and 7-ethoxycoumarin O-deethylase activities of NY-BB were not statistically different from those of FL-BB. cDNA fragments that encoded the open reading frame of voltage-sensitive sodium channel α-subunit genes from the FL-BB and NY-BB populations, respectively, were obtained by homology probing polymerase chain reaction (PCR) and sequenced. Sequence alignment of the internal and 5′ and 3′ rapid amplification of cDNA ends (RACE) fragments generated a 6,500-bp cDNA sequence contig, which was composed of a 6,084-bp open reading frame (ORF) encoding 2,027 amino acid residues and 186-bp 5′ and 230-bp 3′ untranslated regions (5′ and 3′ UTRs, respectively). Sequence comparisons of the open reading frames of the α-subunit genes identified two point mutations (V419L and L925I) that were presented only in the NY-BB population. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly (Bemisia tabaci). V419L, located in the IS6 transmembrane segment, is a novel mutation. A Val to Met mutation at the corresponding position of the bed bug V419, however, has been identified in the tobacco budworm as a kdr-type mutation. This evidence suggests that the two mutations are likely the major resistance-causing mutations in the deltamethrin-resistant NY-BB through a knockdown-type nerve insensitivity mechanism.

Cloning and sequencing of a cDNA encoding acetylcholinesterase in colorado potato beetle, Leptinotarsa decemlineata (say)

A cDNA encoding acetylcholinesterase (AChE, EC 1.1.1.7) was cloned from a cDNA library constructed from an insecticide-susceptible strain of Colorado potato beetle, Leptinotarsa decemlineata (Say). The complete amino acid sequence of AChE deduced from the cDNA consisted of 29 residues for the putative signal peptide and 600 residues for the mature protein with a predicted molecular weight of 67,994. Northern blot analysis of poly(A) RNA showed an approx 13.1-kb transcript. The mature protein sequence had 57 and 61% of amino acid residues identical to those of Drosophila melanogaster and Anopheles stephensi, respectively, and produced a remarkably similar hydropathy profile when compared to those of the two dipterous species. The three residues (Ser, Glu and His) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds were completely conserved when compared to the other seven AChEs from a broad range of animal species reported to date. Other properties of the deduced protein of AChE, including molecular weight and amino acid composition, agreed well with those of a previously reported study on the purified AChE from the same insect species. All these features firmly established that the cloned cDNA encodes AChE in Colorado potato beetle.

Pyrethroid action on calcium channels: neurotoxicological implications

Actions of cismethrin versus deltamethrin were compared using two functional attributes of rat brain synaptosomes. Both pyrethroids increased calcium influx but only deltamethrin increased Ca2+-dependent neurotransmitter release following K+-stimulated depolarization. The action of deltamethrin was stereospecific, concentration-dependent, and blocked by ω-conotoxin GVIA. These findings delineate a separate action for deltamethrin and implicate N-type rat brain Cav2.2xa0voltage-sensitive calcium channels (VSCC) as target sites that are consistent with the in vivo release of neurotransmitter caused by deltamethrin. Deltamethrin (10−7xa0M) reduced the peak current (approx. −47%) of heterologously expressed wild type Cav2.2 in a stereospecific manner. Mutation of threonine 422 to glutamic acid (T422E) in the α1-subunit results in a channel that functions as if it were permanently phosphorylated. Deltamethrin now increased peak current (approx. +49%) of T422E Cav2.2 in a stereospecific manner. Collectively, these results substantiate that Cav2.2 is directly modified by deltamethrin but the resulting perturbation is dependent upon the phosphorylation state of Cav2.2. Our findings may provide a partial explanation for the different toxic syndromes produced by these structurally-distinct pyrethroids.

Comparison of the humoral and cellular immune responses between body and head lice following bacterial challenge

The differences in the immune response between body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, were investigated initially by measuring the proliferation rates of two model bacteria, a Gram-positive Staphylococcus aureus and a Gram-negative Escherichia coli, following challenge by injection. Body lice showed a significantly reduced immune response compared to head lice particularly to E. coli at the early stage of the immune challenge. Annotation of the body louse genome identified substantially fewer immune-related genes compared with other insects. Nevertheless, all required genetic components of the major immune pathways, except for the immune deficiency (Imd) pathway, are still retained in the body louse genome. Transcriptional profiling of representative genes involved in the humoral immune response, following bacterial challenge, revealed that both body and head lice, regardless of their developmental stages, exhibited an increased immune response to S. aureus but little to E. coli. Head lice, however, exhibited a significantly higher phagocytotic activity against E. coli than body lice, whereas the phagocytosis against S. aureus differed only slightly between body andxa0headxa0lice. These findings suggest that the greater immune response in head lice against E. coli is largelyxa0due to enhanced phagocytosis and not due to differences in the humoral immune response. The reduced phagocytotic activity in body lice could be responsible, in part, for their increased vector competence.

Role of ion channels and intraterminal calcium homeostasis in the action of deltamethrin at presynaptic nerve terminals

Using a continuous perfusion system, synaptosomes prepared from rat brain released [3H]norepinephrine in a Ca2+-dependent manner when pulse depolarized by briefly elevating external potassium concentrations. Tetrodotoxin (10(-7) M), a sodium channel blocker, inhibited 48% of this pulsed release, and D595 (10(-5) M), a phenethylamine-type calcium channel blocker, inhibited 21%. In combination, these two specific ion channel antagonists appear to function independently of each other in an additive fashion. Addition of deltamethrin to this preparation resulted in an enhanced release of [3H]norepinephrine which occurred in a biphasic fashion. At 10(-7) M, deltamethrin produced a 42% enhancement in the first or initial peak of [3H]norepinephrine release and a 100% enhancement in the second or tailing peak. Addition of deltamethrin to tetrodotoxin-pretreated synaptosomes resulted in a net 37% enhancement of the initial peak release and a net increase of 277% in the tailing peak. Addition of deltamethrin to D595-pretreated synaptosomes produced no significant effect on enhanced [3H]norepinephrine release from either peak. Since tetrodotoxin is a specific sodium channel blocker, deltamethrin may be enhancing [3H]norepinephrine release by increasing the uptake of Ca2 via other voltage-gated channels (e.g. calcium) or exchange mechanisms in addition to its action at voltage-gated sodium channels. To determine whether deltamethrin may also have an effect on intraterminal Ca2+ homeostasis, external Ca2+ was replaced with Ba2+ and synaptosomes were depolarized with pentylenetetrazole (PTZ). At 10(-5) M, deltamethrin produced a 66% increase in neurotransmitter release over that produced by PTZ alone. An estimated EC50 value of deltamethrin for PTZ-induced release was calculated to be 2.4 x 10(-10) M.

A new ivermectin formulation topically kills permethrin-resistant human head lice (Anoplura: Pediculidae).

Abstract This study examines the effectiveness of a new ivermectin formulation for the topical treatment of the human head louse, Pediculus humanus capitis De Geer (Anoplura: Pediculidae). Permethrin-resistant lice originally obtained from south Florida and maintained on an in vitro rearing system were 100% susceptible to ivermectin formulations by using a semiclinical hair tuft bioassay. The formulation was 100% effective at killing lice using 1, 0.5, and 0.25% ivermectin concentrations after 10-min exposures. As judged by the lethal time (LT)50 and LT95 values, 0.5% formulated ivermectin was 3.8 and 3.2 times faster at killing lice, respectively, than 0.5% nonformulated ivermectin, indicating that the formulation may facilitate the penetration of ivermectin into the louse. The hair tuft-based bioassay in conjunction with the in vitro rearing system provides a standardized method to assess the comparative efficacy of pediculicide formulations in a reproducible format that mimics the exposure scenario that occurs on the human scalp.

Determination of knockdown resistance allele frequencies in global human head louse populations using the serial invasive signal amplification reaction.

BACKGROUNDnPediculosis is the most prevalent parasitic infestation of humans. Resistance to pyrethrin- and pyrethroid-based pediculicides is due to knockdown (kdr)-type point mutations in the voltage-sensitive sodium channel alpha-subunit gene. Early detection of resistance is crucial for the selection of effective management strategies.nnnRESULTSnKdr allele frequencies of lice from 14 countries were determined using the serial invasive signal amplification reaction. Lice collected from Uruguay, the United Kingdom and Australia had kdr allele frequencies of 100%, while lice from Ecuador, Papua New Guinea, South Korea and Thailand had kdr allele frequencies of 0%. The remaining seven countries investigated, including seven US populations, two Argentinian populations and populations from Brazil, Denmark, Czech Republic, Egypt and Israel, displayed variable kdr allele frequencies, ranging from 11 to 97%.nnnCONCLUSIONnThe newly developed and validated SISAR method is suitable for accurate monitoring of kdr allele frequencies in head lice. Proactive management is needed where kdr-type resistance is not yet saturated. Based on sodium channel insensitivity and its occurrence in louse populations resistant to pyrethrin- and pyrethroid-based pediculicides, the T917I mutation appears to be a key marker for resistance. Results from the Egyptian population, however, indicate that phenotypic resistance of lice with single or double mutations (M815I and/or L920F) should also be determined.

Advances in the Mode of Action of Pyrethroids

The ability to clone, express, and electrophysiologically measure currents carried by voltage-gated ion channels has allowed a detailed assessment of the action of pyrethroids on various target proteins.Recently, the heterologous expression of various rat brain voltage-gated sodium channel isoforms in Xenopus laevis oocytes has determined a wide range of sensitivities to the pyrethroids, with some channels virtually insensitive and others highly sensitive. Furthermore, some isoforms show selective sensitivity to certain pyrethroids and this selectivity can be altered in a state-dependent manner. Additionally, some rat brain isoforms are apparently more sensitive to pyrethroids than the corresponding human isoform. These finding may have significant relevance in judging the merit and value of assessing the risk of pyrethroid exposures to humans using toxicological studies done in rat.Other target sites for certain pyrethroids include the voltage-gated calcium and chloride channels. Of particular interest is the increased effect of Type II pyrethroids on certain phosphoforms of the N-type Ca(v)2.2 calcium channel following post-translational modification and its relationship to enhanced neurotransmitter release seen in vivo.Lastly, parallel neurobehavioral and mechanistic studies on three target sites suggest that a fundamental difference exists between the action of Types I and II pyrethroids, both on a functional and molecular level. These differences should be considered in any future risk evaluation of the pyrethroids.

G-protein modulators alter the swimming behavior and calcium influx of Paramecium tetraurelia

Abstract To assess the potential role of G-proteins in chemokinesis, Paramecium tetraurelia was pre-incubated with the G-protein modulator pertussis toxin. Pertussis toxin pretreatment significantly reduced Paramecium chemoattraction to sodium acetate and ammonium chloride in T-maze behavioral assays and depressed the frequency of avoidance reactions, indicating that heterotrimeric G-proteins may be involved with the motility response. To determine whether G-proteins exert their effect via the ciliary voltage-sensitive calcium channel, we examined responses of P. tetraurelia to the potent voltage-sensitive calcium channel agonist, deltamethrin. Pertussis toxin preincubation significantly reduced the toxic effects of deltamethrin exposure as determined by survival under depolarizing conditions and reduced the duration of backward swimming episodes in behavioral bioassays. Furthermore, non-hydrolyzable analogs of guanine nucleotides altered deltamethrin-stimulated calcium influx via calcium channels in isolated ciliary vesicles. Heterotrimeric G-protein subunits were subsequently detected in ciliary vesicles of P. tetraurelia by antibodies produced against Gα and Gβ subunits, and by 32P-ADP-ribosylation, indicating that proteins of the appropriate molecular weight are the target of pertussis toxin in these vesicles. These findings provide additional evidence that heterotrimeric G-proteins are associated with ciliary vesicles and that they play a role in the modulation of swimming behavior and the toxic action of deltamethrin in Paramecium.

Ivermectin Acts as a Posteclosion Nymphicide by Reducing Blood Feeding of Human Head Lice (Anoplura: Pediculidae) That Hatched From Treated Eggs

ABSTRACT n The 0.5% ivermectin topical cream formulation was not directly ovicidal to treated eggs of head lice, as hatchability was not decreased. Nevertheless, the percent of hatched lice from treated eggs that took a blood meal significantly decreased (80–95%) compared with lice that hatched from untreated eggs and all treated lice died within 48 h of hatching, including those that fed. Dilutions of ivermectin formulation of 0.15 and 0.2 µg/ml, which were topically applied to 0–8 d old eggs, were not lethal to lice at 24 h posteclosion. However, 9 and 16% less lice fed when hatched from these treated eggs, respectively. Total [3H] inulin ingested by untreated first instars significantly increased over a 48 h feeding interval but was significantly less in instars that hatched from eggs receiving the 0.15 (36% less) and 0.2 (55% less) µg/ml ivermectin treatments compared with placebo. The reduced feeding that occurred after the 0.15 and 0.2 µg/ml ivermectin treatments occurred in the absence of mortality and suggests a unique mode of action of ivermectin on feeding that is separate from the mode of action of ivermectin leading to mortality. Failure of hatched instars to take a blood meal after egg treatments with formulated ivermectin is likely responsible for its action as a posteclosion nymphicide.