Cloning and sequencing of a putative acetylcholinesterase cDNA from Boophilus microplus (Acari: Ixodidae)

Using a strategy based on degenerate primers derived from acetylcholinesterase (AChE) from other species, we cloned and sequenced a putative AChE cDNA from the southern cattle tick, Boophilus microplus (Canestrini). The sequence has a high degree of homology to sequences of AChE from other species reported in the GenBank. The open reading frame of 1,689 bp, corresponding to a deduced sequence of 563 amino acids, has conserved regions and features shared by the AChE family, necessary for its catalytic activity. No differences were found in the putative cDNA sequences from organophosphorus acaricide (OP) resistant and susceptible strains. The results suggest that this putative AChE gene is not involved in resistance to OP compounds as a mutated gene in the resistant strain studied. However, differences were detected, with a probe derived from this cDNA, in DNA fragments after digestion of genomic DNA from different strains with restriction nucleases. This indicates polymorphism in this gene in B. microplus.     

Molecular analysis of a para sodium channel gene from pyrethroid-resistant head lice, Pediculus humanus capitis (Anoplura: Pediculidae)

The problem of pyrethroid-resistance in head lice, Pediculus humanus capitis (De Geer), is growing worldwide, and an insensitive sodium channel is suspected as the major mechanism of this resistance. We sequenced an open reading frame (ORF) encoding for the para-orthologous sodium channel from an insecticide-susceptible strain of the body louse, Pediculus humanus humanus (L.), based on conserved peptide sequences and a known partial gene sequence. Phenothrin-susceptible and -resistant head louse colonies from Japanese were individually analyzed for point mutations of the sodium channel cDNA; susceptible head and body lice differed in double homozygous synonymous substitutions. The resistant head lice shared 23 base substitutions homozygously, in which four resulted in amino acid substitutions: D11E in the N-terminal inner-membrane segment; M850T in the outer-membrane loop between segments four and five of domain II; T952I and L955 F in the trans-membrane segment five of domain II. The latter two substitutions coincided with those of pyrethroid-resistant head lice in the U.S. and U.K. (Lee et al. 2000), within the available published information on the peptide sequences. The potential mechanisms of head louse pyrethroid-resistance are discussed based on the four structural changes of the target molecule.     

Molecular survey of pyrethroid resistance mechanisms in Mexican field populations of Rhipicephalus (Boophilus) microplus

Susceptibility to synthetic pyrethroids (SP′s) and the role of two major resistance mechanisms were evaluated in Mexican Rhipicephalus microplus tick populations. Larval packet test (LPT), knock-down (kdr) PCR allele-specific assay (PASA) and esterase activity assays were conducted in tick populations for cypermethrin, flumethrin and deltamethrin. Esterase activity did not have a significant correlation with SP′s resistance. However a significant correlation (p < 0.01) was found between the presence of the sodium channel mutation, and resistance to SP′s as measured by PASA and LPT respectively. Just over half the populations (16/28) were cross-resistant to flumethrin, deltamethrin and cypermethrine, 21.4% of the samples (6/28) were susceptible to all of the three pyrethroids 10.7 of the samples (3/28) were resistant to flumethrin, 3.4 of the samples (1/28) were resistant to deltamethrin only and 7.1% (2/28) were resistant to flumethrin and deltamethrin. The presence of the kdr mutation correlates with resistance to the SP′s as a class. Target site insensitivity is the major mechanism of resistance to SP′s in Mexican R. microplus field strains, involving the presence of a sodium channel mutation, however, esterase-based, other mutations or combination of mechanisms can also occur.     

Detection of knockdown resistance mutations in Anopheles sacharovi (Diptera: Culicidae) and genetic distance with Anopheles gambiae (Diptera: Culicidae) using cDNA sequencing of the voltage-gated sodium channel gene

The knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene (VGSCG), an important resistance mechanism against pyrethroids, was studied in Anopheles sacharovi Favre. It was found that the specific primers Agd1 and Agd2 used for polymerase chain reaction (PCR) amplification of Anopheles gambiae Giles VGSCG also amplified this genomic region in An. sacharovi. Comparison of the IIs4-IIs6 domain segments of the gene indicated 70% nucleotides common to both species and a genetic distance of 0.255 between them. Four different samples of pyrethroid-resistant An. sacharovi produced three types of amino acid, serine (TCG),leucine (TTG),and phenylalanine (TTT) at the kdr mutation point, whereas only two kdr mutations, leucine to phenylalanine and leucine to serine, occur in An. gambiae.     

Posttreatment with sodium arsenite alters the mutational spectrum induced by ultraviolet light irradiation in Chinese hamster ovary cells.

Arsenic, a potent carcinogen, fails to induce gene mutations in mammalian cells. However, posttreatment of ultraviolet light (UV) irradiated cells with sodium arsenite synergistically enhances the mutation frequency on the hypoxanthine (guanine) phosphoribosyltransferase locus. To investigate the molecular mechanism of the comutagenic effects of sodium arsenite, we characterized the alterations of nucleotide sequences in 30 UV-induced and 39 sodium arsenite enhanced hprt mutants from Chinese hamster ovary K1 cells by direct sequencing of mRNA-PCR amplified cDNA. The majority of sequence alterations derived from UV irradiation (80%) and from sodium arsenite posttreatment (70%) were single base substitutions. UV irradiation induced all types of base substitutions. Among them, 57% were transversions. The frequency of transversions increased to 70% in sodium arsenite enhanced mutants. While base substitutions observed in UV-induced mutants were evenly distributed along with the whole coding region, exons 3 and 8 were most frequently mutated in sodium arsenite enhanced mutants. Sodium arsenite posttreatment did not alter the strand bias for mutation induction, i.e., 73% and 78%, of the mutations were located on the non-transcribed strand in UV-induced and sodium arsenite enhanced mutants, respectively. In contrast to UV-induced mutations, bases at the 5' position of TT and the 3' position of CT sequences were the most frequent mutation sites observed in sodium arsenite enhanced mutants. We hypothesize that sodium arsenite may interfere with the process of mutation fixation of TT and CT dimers during DNA replication.     

Mapping and sequencing of the canine NRAMP1 gene and identification of mutations in leishmaniasis-susceptible dogs

The NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control of intraphagosomal replication of parasites and in macrophage activation. It has been described in mice as the determinant of natural resistance or susceptibility to infection with antigenically unrelated pathogens, including LEISHMANIA: Our aims were to sequence and map the canine Slc11a1 gene and to identify mutations that may be associated with resistance or susceptibility to Leishmania infection. The canine Slc11a1 gene has been mapped to dog chromosome CFA37 and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellite in intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1 proteins of different mammalian species. A case-control study with 33 resistant and 84 susceptible dogs showed an association between allele 145 of the microsatellite and susceptible dogs. Sequence variant analysis was performed by direct sequencing of the cDNA and the promoter region of four unrelated beagles experimentally infected with Leishmania infantum to search for possible functional mutations. Two of the dogs were classified as susceptible and the other two were classified as resistant based on their immune responses. Two important mutations were found in susceptible dogs: a G-rich region in the promoter that was common to both animals and a complete deletion of exon 11, which encodes the consensus transport motif of the protein, in the unique susceptible dog that needed an additional and prolonged treatment to avoid continuous relapses. A study with a larger dog population would be required to prove the association of these sequence variants with disease susceptibility.     

Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations

Congenital indifference to pain (CIP) is a rare condition in which patients have severely impaired pain perception, but are otherwise essentially normal. We identified and collected DNA from individuals from nine families of seven different nationalities in which the affected individuals meet the diagnostic criteria for CIP. Using homozygosity mapping and haplotype sharing methods, we narrowed the CIP locus to chromosome 2q24-q31, a region known to contain a cluster of voltage-gated sodium channel genes. From these prioritized candidate sodium channels, we identified 10 mutations in the SCN9A gene encoding the sodium channel protein Nav1.7. The mutations completely co-segregated with the disease phenotype, and nine of these SCN9A mutations resulted in truncation and loss-of-function of the Nav1.7 channel. These genetic data further support the evidence that Nav1.7 plays an essential role in mediating pain in humans, and that SCN9A mutations identified in multiple different populations underlie CIP.     

Mapping of the RD phenotype of the Nancy strain of coxsackievirus B3.

The RD variants of group B coxsackieviruses differ from their parental strains in having the ability to replicate in a human rhabdomyosarcoma cell line, RD. The nucleotide sequence of the P1 region of the RD variant of coxsackievirus B3 strain Nancy (CB3NRD) was determined by sequencing cloned cDNAs, obtained by PCR amplification. A comparison between the established nucleotide sequence and that of the P1 region from the parental virus revealed 12 point mutations which corresponded to six amino acid replacements. To identify if the P1 region is responsible for the phenotype of CB3NRD, a chimeric virus was constructed, using an infectious cDNA clone of CB3. The P1 region of the infectious cDNA was replaced by cDNA fragments from CB3N (parental strain Nancy) or CB3NRD and the resulting recombinants were assayed for their ability to infect and replicate in RD cells. The results showed that the RD phenotype of CB3NRD maps in the P1 region. Furthermore, a chimera which only contained the 5' part of the P1 region derived from CB3NRD and the remaining P1 sequence from CB3N was able to replicate in RD cells, suggesting that the VP2 polypeptide contains at least one determinant for the RD phenotype.     

Mode of action for natural products isolated from essential oils of two trees is different from available mosquito adulticides

Insecticidal properties of natural products may present alternatives to the use of synthetic molecule pesticides that are of diminishing effectiveness due to resistance. Three compounds, thymoquinone, nootkatone, and carvacrol, components of Alaska yellow cedar, Chamaecyyparis nootkatensis (D. Don) Spach, and incense cedar, Calocedrus decurrens (Torr.), essential oils, have been shown to have biological activity against a variety of mosquito and tick species. Although these components act as both repellents and insecticides, how they function in either capacity is unknown. Their use as mosquito control insecticides would be greatly increased if their mode of action is not the same as that of currently used commercial products. This study compared the lethal dosages for nootkatone, carvacrol, and thymoquinone by using colony strains of Anopheles gambiae Giles with known mutations at three different target sites. The altered target sites evaluated were the sodium channel para-locus mutation (L1014 F KDR) that confers permethrin resistance, the ACE-1 gene that confers organophosphate and carbamate resistance, and a gamma-aminobutyric acid receptor mutation of the Rdl locus conferring dieldrin resistance. Significant increases in lethal dose were not observed in any of the mosquito strains for any of the compounds tested compared with the doses required of chemicals with known modes of action at the mutated sites. Although the mode of action was not determined, this screening study indicates that none of these compounds interact at the target sites represented in the test mosquito strains. These compounds represent a different mode of action than existing chemicals currently used in mosquito control.     

Two new mutations in the dihydropteridine reductase gene in patients with tetrahydrobiopterin deficiency.

Two new mutations have been identified within the dihydropteridine reductase (DHPR) gene in two patients with DHPR deficiency. The total coding sequence of the cDNA has been screened by chemical cleavage of mismatch in both patients and selected portions of the cDNA have been sequenced. The first mutation identified causes a glycine to aspartic acid substitution at codon 23 and seems particularly frequent in Mediterranean patients. Its occurrence within a glycine string common to the amino-terminal region in NADH dependent enzymes suggests a possible causal mechanism for the defect. The second change involves a tryptophan to glycine substitution at codon 108 and is carried by both alleles in the second patient. It occurs in a motif which shows similarities with a region of dihydrofolate reductase (DHFR) and is highly conserved within different animal species.     

R86Q, a mutation in BmAChE3 yielding a Rhipicephalus microplus organophosphate-insensitive acetylcholinesterase

Mutations were identified in the cDNA sequence encoding the acetylcholinesterase BmAChE3 in strains of Rhipicephalus (Boophilus) microplus (Canestrini) resistant or susceptible to organophosphate (OP) acaricide. The mutation that occurred most frequently in the OP-resistant San Román strain resulted in a substitution of glutamine (Q) for arginine (R) at position 86 in BmAChE3 (position 66 in mature BmAChE). Clones containing the mutant and wild-type cDNA sequences were expressed in the baculovirus system. Enzyme kinetics of recombinant BmAChE3 containing or lacking the R86Q mutation demonstrated that the R86Q mutation increased substrate affinity and conferred insensitivity to paraoxon inhibition. This is the first demonstration of a mutation in a gene encoding an ixodid acetylcholinesterase resulting in OP insensitivity. A restriction fragment length polymorphism assay was developed and used to diagnose the frequency of the R86Q mutation in BmAChE3 genomic DNA from seven laboratory-colonized strains. Use of the R86Q diagnostic assay detected an increased frequency of the R86Q mutation in OP-resistant tick strains compared with that of OP-susceptible strains; however, the R86Q mutation was also present in OP-susceptible strains at unexpectedly high frequency. Because the R86Q mutation generates an OP-resistant enzyme in vitro and it is present at an elevated frequency in laboratory strains selected for OP resistance, we conclude that the data are consistent with a potential role for BmAChE3 in development of OP resistance; however, because the R86Q mutation has a high frequency in susceptible strains, the R86Q mutation alone is insufficient to generate the OP-resistant phenotype at the organismal level. There are likely to be additional mutations in BmAChE3, mutations in additional acetylcholinesterase genes, or additional resistance mechanisms (e.g., oxidative metabolism) that contribute to expression of the OP-resistant phenotype.     

Cutaneous basophil-associated resistance to ectoparasites (ticks). I. Transfer with immune serum or immune cells.

Immune resistance experiments were carried out in guinea-pigs employing two tick species that as adults are ectoparasites of cattle (Ixodes holocyclus and Rhipicephalus appendiculatus). These studies showed that susceptibility of non-immune guinea-pigs to infestation with tick larvae varies according to the species of tick and the strain of guinea-pig. With both tick species, greater than 90% acquired resistance was achieved in several guinea-pig strains. Immune resistance was evident within a week following primary infestation and lasted up to 9 months following a single sensitizing exposure to tick feeding. The strength and duration of resistance was influenced strongly by the size of the initial sensitizing dose. Immune resistance was readily transferred to naive recipients by intravenous administration of either peritoneal exudate cells or immune serum from donors sensitized by a single prior infestation with ticks. Doses of serum as small as 0.5 ml transferred resistance. These studies demonstrate that both sensitized cells and immune serum factors contribute significantly to acquired host resistance to ticks that as adults are ectoparasites of cattle.     

Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: isolation, structural characterization and tissue expression profile

Antimicrobial peptides (AMPs) are components of the immune system of both vertebrate and invertebrate animals. This study describes the isolation, primary structure, cDNA cloning, and tissue expression profile of two cysteine-rich AMPs from the hemolymph of the cattle tick Boophilus microplus. A 10,204 Da polypeptide, with six cysteine residues and no sequence similarity to any known molecule, was isolated from the cell-free hemolymph. Because of its sequence originality, this peptide was named microplusin. The second AMP was isolated from the hemocytes of B. microplus. This peptide, with a molecular mass of 4285 Da and six cysteines, is a defensin with similarity to the insect defensin family members. The cDNA cloning established that microplusin is synthesized as a prepeptide while the tick defensin is synthesized as a prepromolecule. Interestingly, despite the fact that microplusin and defensin have been isolated from different compartments, their gene expression was found to have similar tissue distribution.     

Use of the polymerase chain reaction to investigate the dynamics of pyrethroid resistance in Haematobia irritans irritans (Diptera: Muscidae)

A field study was conducted from 1991 through 1997 to evaluate the use of pyrethroid and organophosphate (OP) ear tags, alternated yearly, for the control of a pyrethroid resistant horn fly, Haematobia irritans (L.), population in Louisiana. Fly resistance was monitored by weekly fly counts, filter paper bioassays and diagnostic polymerase chain reaction (PCR) assays for the presence of pyrethroid resistance-associated mutations in the sodium channel gene coding region. Fly control in the first study year was poor, as pyrethroid ear tags were effective for only 7 wk. The following year, OP ear tags provided 15 wk of fly control. However, in all subsequent years, fly control was poor with both types of ear tags. The PCR assays showed that there were very few female flies homozygous for the pyrethroid susceptible sodium channel allele, never rising above 10% of the total females in the population. A fitness cost appeared to be associated with the pyrethroid resistant allele, as the resistant form was selected against in the absence of the pyrethroid ear tags. Despite this selection in favor of the susceptible allele and the annual alternation of pyrethroid and OP ear tags, the percentage of homozygous susceptible flies never reached over 19% of the population, resistant alleles of the sodium channel remained at high levels in the population, and horn fly control on cattle with either type of tag quickly became minimal.     

Application of representational difference analysis to identify sequence tags expressed by Metarhizium anisopliae during the infection process of the tick Boophilus microplus cuticle

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of plagues, including insects and acari. To identify genes involved in the infection process, representational difference analysis was performed using cDNA generated from germinated conidia of M. anisopliae in the tick Boophilus microplus cuticle, and cDNA generated during fungal growth in glucose-rich medium. Sequence determination of approximately 135 clones and comparison analysis using public databases led to the identification of 34 sequences and 14 expressed sequence tags with known orthologs. As expected, almost all identified sequences showed significant similarity to other fungal genes. The diversity of gene clusters found reflects the participation of several proteins in the early infection process of M. anisopliae in the cattle tick B. microplus.     

The genomic structure of the human skeletal muscle sodium channel gene.

Electrical excitability of neurons and muscle cells reflects the actions of a family of structurally related sodium channels. Mutations in the adult skeletal muscle sodium channel have been associated with the inherited neuromuscular disorders paramyotonia congenita (PMC) and hyperkalemic periodic paralysis (HPP). We have deciphered the entire genomic structure of the human skeletal muscle sodium channel gene and developed a restriction map of the locus. SCN4A consists of 24 exons spanning 35 kb of distance on chromosome 17q. We describe the sequence of all intron/exon boundaries, the presence of several polymorphisms in the coding sequence, and the locations within introns of two dinucleotide repeat polymorphisms. This is the first sodium channel for which the entire genomic structure has been resolved. The organization of the SCN4A exons relative to the proposed protein structure is presented and represents a foundation for functional and evolutionary comparisons of sodium channels. Knowledge of the exon structure and flanking intron sequences for SCN4A will permit a systematic search for mutations in PMC and HPP.     

An additional family of VH sequences in the channel catfish.

A heavy chain variable region cDNA sequence of the channel catfish (Ictalurus punctatus) prototypical for a new VH family (approximately 20 or more members) is presented. The nucleotide and inferred amino acid sequences differ by 32-52% and 41-68%, respectively, from those for the already described catfish VH families.     

Detection of a knockdown resistance mutation associated with permethrin resistance in the body louse Pediculus humanus corporis by use of melting curve analysis genotyping

Louse-borne diseases are prevalent in the homeless, and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based on real-time PCR using hybridization probes to detect the M815I-T917I-L920F knockdown resistance (kdr) mutation in the paraorthologous voltage-sensitive sodium channel (VSSC) α subunit gene, which is associated with permethrin resistance. The 908-bp DNA fragment of the VSSC gene, encoding the α subunit of the sodium channel and encompassing the three mutation sites, was PCR sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping, which had a calculated sensitivity of 100% for the M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for T917I. Melting curve analysis genotyping is a fast, sensitive, and specific tool that is fully compatible with the analysis of a large number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75 min). Thus, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale clinical studies.     

Simultaneous identification of campylobacters and prediction of quinolone resistance by comparative sequence analysis.

Comparative sequence analysis of a 30-bp segment in the quinolone resistance-determining region of campylobacters not only allows for the detection of base changes associated with resistance but also is a powerful tool for species identification based on silent mutations.