Exome-wide association of deltamethrin resistance in Aedes aegypti from Mexico.

Aedes aegypti is the major vector of a number of arboviruses that cause disease in humans. Without vaccines or pharmaceuticals, pyrethroid insecticides remain the major tool for public health protection. Pyrethroid resistance is now widespread. Replacement substitutions in the voltage-gated sodium channel (vgsc) that reduce the stability of pyrethroid binding account for most of the resistance, but metabolic mechanisms also inactivate pyrethroids. High-throughput sequencing and the A. aegypti L5 annotated physical map has allowed interrogation of the exome for genes and single-nucleotide polymorphisms associated with pyrethroid resistance. We exposed females of A. aegypti from Mexico to a deltamethrin discriminating dose to designate them as resistant (active after 1 h) or susceptible (knocked down with no recovery after 4 h). The vgsc on chromosome 3 had the highest association, followed by genes proximal to vgsc. We identified potential detoxification genes located singly (eg HPX8C) or within clusters in chromosome 2 [three esterase clusters, two of cytochrome P450 monooxygenases (CYP)] and chromosome 3 (one cluster of 16 CYP325 and seven CYP9 genes). Deltamethrin resistance in A. aegypti is associated with mutations in the vgsc gene and a large assortment of genes.

Inhibition of bone resorption by the cathepsin K inhibitor odanacatib is fully reversible.

The cathepsin K (CatK) inhibitor odanacatib (ODN) is currently being developed for the treatment of osteoporosis. In clinical trials, efficacy and resolution of effect of ODN treatment on bone turnover biomarkers and accrued bone mass have been demonstrated. Here, we examine the effects of continuing treatment and discontinuation of ODN versus alendronate (ALN) on osteoclast (OC) function. First, accessibility and reversible engagement of active CatK in intracellular vesicles and resorption lacunae of actively resorbing OCs were demonstrated by the selective and reversible CatK inhibitors, BODIPY-L-226 (IC50=39nM) and L-873,724 (IC50=0.5nM). Next, mature human OCs on bone slices were treated with vehicle, ODN, or ALN for 2days, followed by either continuing with the same treatment, or replacement of the inhibitors by vehicle for additional times as specified per experimental conditions. Maintaining OCs on ODN or ALN significantly reduced CTx-I release compared to vehicle controls. However, only the treatment of OCs with ODN resulted in the formation of small shallow discrete resorption pits, retention of intracellular vesicles enriched with CatK and other lysosomal enzymes, increase in 1-CTP release and number of TRAP(+) OCs. Upon discontinuation of ODN treatment, OCs rapidly resumed bone resorption activity, as demonstrated by a return of OC functional markers (CTx-I, 1-CTP), cell number and size, morphology and number of resorption pits, and vesicular secretion of CatK toward the respective vehicle levels. As expected, discontinuation of ALN did not reverse the treatment-related inhibition of OC activity in the time frame of the experiment. In summary, this study demonstrated rapid kinetics of inhibition and reversibility of the effects of ODN on OC bone resorption, that differentiated the cellular mechanism of CatK inhibition from that of the bisphosphate antiresorptive ALN.

Differential transcription profiles in Aedes aegypti detoxification genes after temephos selection.

The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti 'Detox Chip' microarray during five generations of temephos selection. We selected for temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione-S-transferase (eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of temephos selection. While expression of many carboxyl/cholinesterase esterase (CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase (CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione-S-transferase (GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to temephos. We show that temephos-selected strains show no cross resistance to permethrin and that genes associated with temephos selection are largely independent of those selected with permethrin in a previous study.

Proof of concept for a novel insecticide bioassay based on sugar feeding by adult Aedes aegypti (Stegomyia aegypti).

Aedes aegypti L. (Stegomyia aegypti) (Diptera: Culicidae) is the principal vector of dengue and yellow fever viruses in tropical and subtropical regions of the world. Disease management is largely based on mosquito control achieved by insecticides applied to interior resting surfaces and through space sprays. Population monitoring to detect insecticide resistance is a significant component of integrated disease management programmes. We developed a bioassay method for assessing insecticide susceptibility based on the feeding activity of mosquitoes on plant sugars. Our prototype sugar-insecticide feeding bioassay system was composed of inexpensive, disposable components, contained minimal volumes of insecticide, and was compact and highly transportable. Individual mosquitoes were assayed in a plastic cup that contained a sucrose-permethrin solution. Trypan blue dye was added to create a visual marker in the mosquito's abdomen for ingested sucrose-permethrin solution. Blue faecal spots provided further evidence of solution ingestion. With the sugar-insecticide feeding bioassay, the permethrin susceptibility of Ae. aegypti females from two field-collected strains was characterized by probit analysis of dosage-response data. The field strains were also tested by forced contact of females with permethrin residues on filter paper. Dosage-response patterns were similar, indicating that the sugar-insecticide feeding bioassay had appropriately characterized the permethrin susceptibility of the two strains.

The effect of ivermectin in seven strains of Aedes aegypti (Diptera: Culicidae) including a genetically diverse laboratory strain and three permethrin resistant strains.

Seven different strains of Aedes aegypti (L.), including a genetically diverse laboratory strain, three laboratory-selected permethrin-resistant strains, a standard reference strain, and two recently colonized strains were fed on human blood containing various concentrations of ivermectin. Ivermectin reduced adult survival, fecundity, and hatch rate of eggs laid by ivermectin-treated adults in all seven strains. The LC50 of ivermectin for adults and the concentration that prevented 50% of eggs from hatching was calculated for all strains. Considerable variation in adult survival after an ivermectin-bloodmeal occurred among strains, and all three permethrin-resistant strains were significantly less susceptible to ivermectin than the standard reference strain. The hatch rate after an ivermectin bloodmeal was less variable among strains, and only one of the permethrin-resistant strains differed significantly from the standard reference strain. Our studies suggest that ivermectin induces adult mortality and decreases the hatch rate of eggs through different mechanisms. A correlation analysis of log-transformed LC50 among strains suggests that permethrin and ivermectin cross-resistance may occur.

Evidence of multiple chromosomal inversions in Aedes aegypti formosus from Senegal.

Chromosomal inversions are prevalent in mosquito species but polytene chromosomes are difficult to prepare and visualize in members of the tribe Aedinii and thus there exists only indirect evidence of inversions. We constructed an F(1) intercross family using a P(1) female from a laboratory strain of Aedes aegypti aegypti (Aaa) and a P(1) male Aedes aegypti formosus (Aaf) from a strain collected from south-eastern Senegal. Recombination rates in the F(2) offspring were severely reduced and genotype ratios suggested a deleterious recessive allele on chromosome 3. The F(2) linkage map was incongruent in most respects with the established map for Aaa. Furthermore, no increased recombination was detected in F(5) offspring. Recombination rates and gene order were consistent with the presence in Aaf of at least four large inversions on chromosome 1, a single small inversion on chromosome 2 and three inversions on chromosome 3.

Quantitative trait loci determining autogeny and body size in the Asian tiger mosquito (Aedes albopictus).

The majority of mosquito species require a blood meal to stimulate vitellogenesis and subsequent oviposition (anautogeny), but some autogenous individuals complete their first ovarian cycle without a blood meal. Autogeny may be facultative or obligatory. In this study, we selected for an autogenous strain in the Asian tiger mosquito Aedes albopictus and examined an F(1) intercross population for quantitative trait loci (QTL) determining the autogeny trait as well as wing length as a proxy for body size. Using composite interval mapping, we identified four QTL for each trait and observed considerable overlap in genome positions between each QTL for autogeny (follicle size) and wing length. Most QTL were minor in magnitude, individually explaining <10% of the phenotypic variation. Alleles from the autogenous parent generally showed a dominance or overdominance effect on both phenotypes. Strong genetic and phenotypic correlations indicate that autogeny and wing length are determined by up to four clusters of tightly linked genes or the potential pleiotropic effects of single genes. Although females from the autogenous strain produced approximately fivefold more eggs following a blood meal than through autogeny, we suggest that the maintenance of alleles for autogeny in natural populations is likely due to balancing selection. Autogeny should be favored under conditions of limited host availability for blood feeding or increased defensive behavior by the host and adequate larval nutrition. Correlation between autogeny and body size may reflect an increased ability for larger females to accumulate sufficient nutrient reserves to support oogenesis without the requirement for a blood meal.

A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti.

Pyrethroids are commonly used as mosquito adulticides and evolution of resistance to these compounds is a major threat to public health. 'Knockdown resistance' to pyrethroids (kdr) is frequently caused by nonsynonymous mutations in the voltage-gated sodium channel transmembrane protein (para) that reduce pyrethroid binding. Early detection of kdr is critical to the development of resistance management strategies in mosquitoes including Aedes aegypti, the most prevalent vector of dengue and yellow fever viruses. Brengues et al. described seven novel mutations in hydrophobic segment 6 of domain II of para in Ae. aegypti. Assays on larvae from strains bearing these mutations indicated reduced nerve sensitivity to permethrin inhibition. Two of these occurred in codons Iso1011 and Val1016 in exons 20 and 21 respectively. A transition in the third position of Iso1011 encoded a Met1011 replacement and a transversion in the second position of Val1016 encoded a Gly1016 replacement. We have screened this same region in 1318 mosquitoes in 32 additional strains; 30 from throughout Latin America. While the Gly1016 allele was never detected in Latin America, we found two new mutations in these same codons. A transition in the first position of codon 1011 encodes a Val replacement while a transition in the first position of codon 1016 encodes an Iso replacement. We developed PCR assays for these four mutations that can be read either on an agarose gel or as a melting curve. Selection experiments, one with deltamethrin on a field strain from Santiago de Cuba and another with permethrin on a strain from Isla Mujeres, Mexico rapidly increased the frequency of the Iso1016 allele. Bioassays of F(3) offspring arising from permethrin susceptible Val1016 homozygous parents and permethrin resistant Iso1016 homozygous parents show that Iso1016 segregates as a recessive allele in conferring kdr. Analysis of segregation between alleles at the 1011 and 1016 codons in the F(3) showed a high rate of recombination even though the two codons are only separated by a ~250 bp intron. The tools and information presented provide a means for early detection and characterization of kdr that is critical to the development of strategies for resistance management.

Affordable assays for genotyping single nucleotide polymorphisms in insects.

Insect genome projects and DNA sequence databases are providing unprecedented amounts of information about variation at specific nucleotides in protein- and RNA-coding genes. Single nucleotide polymorphisms (SNPs) are abundant in all insect species so far examined and are proving useful in population genetics, linkage mapping and marker-assisted selection. A number of studies has already identified SNPs associated with insecticide resistance, especially mutations conferring reduced target site sensitivity. Unfortunately, most modern, high-throughput, automated SNP detection technologies are expensive or require the use of expensive equipment and are therefore not accessible to laboratories on a limited budget or to our colleagues in developing countries. In this review, we provide a chronological and comprehensive list of all SNP methods. We emphasize and explain those techniques in which genotypes can be identified by eye or that only require agarose gel electrophoresis. We provide examples where these techniques have or are currently being applied to insects.

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Dengue 2 genotypes in the state of Oaxaca, Mexico.

To genetically characterize dengue 2 (DEN-2) viruses in Oaxaca, Mexico, the C protein, and a portion of the prM protein genes of 8 isolates from the 2001 DEN epidemic were sequenced. The sequences were compared to those of prototype DEN-2 viruses from various parts of the world. Phylogenetic analysis suggested that the 2001 isolates of DEN-2 were of the American/Asian genotype and were most similar to the Jamaica and Venezuelan isolates MARA3, LARD1996 and LARD1910. Molecular analyses confirmed the origin of the isolates. This study indicates that DEN-2 strains of American/Asian genotype probably from Southeast Asian are circulating in Oaxaca.

Genome size and organization in the blacklegged tick, Ixodes scapularis and the Southern cattle tick, Boophilus microplus.

Genome sizes and the organization of repetitive DNA were determined in the hard ticks Ixodes scapularis and Boophilus microplus using reassociation kinetics. The I. scapularis genome contains approximately 2.15 pg (2.1x10(3) Mbp) of DNA and consists of no foldback (FB), 27% highly repetitive (HR), 39% moderately repetitive (MR), and 34% unique DNA. The B. microplus genome contains 7.5 pg (7.1x10(3) Mbp) DNA, and consists of 0.82% FB, 31% HR, 38% MR, and 30% unique DNA. In both species, repetitive sequences occur in a mixture of long and short period interspersion but most (65-80%) of the DNA follows a pattern of short period interspersion. Genome size and organization in the three tick species so far examined are distinct from other arthropods in having a greater proportion of MR, a lower proportion of unique and HR DNA of very low sequence complexity.

Quantitative trait loci affecting dengue midgut infection barriers in an advanced intercross line of Aedes aegypti.

Quantitative trait loci (QTL) affecting the ability of the Aedes aegypti midgut to become infected with Dengue 2 virus (DEN2) were mapped in the F5 generation of an advanced intercross line (AIL). A strain of Ae. aegypti previously selected for DEN2 susceptibility was crossed to a new strain selected for refractoriness to midgut infection. In P1 and F1 parents and 147 F5 offspring, genotypes at forty-four cDNA loci were analysed. A new sex linked QTL and a second QTL on chromosome II with genotypes subject to balancing selection were detected that condition midgut susceptibility. Alleles at these QTL contributed additively in determining susceptibility and accounted for approximately 24% of the phenotypic variance. Markers associated with a midgut escape barrier were inconsistently supported.

Identification of quantitative trait loci affecting sex determination in the Eastern treehole mosquito (Ochlerotatus triseriatus).

Laboratory colonies of the eastern treehole mosquito (Ochlerotatus triseriatus (Say)) exhibit a consistent female-biased sex ratio. This is unusual among mosquito species, in which heritable sex ratio distortion is usually male biased and mediated by meiotic drive. Quantitative trait loci (QTL) affecting sex were mapped in an F(1) intercross to better understand the genetics underlying this female bias. In P(1) and F(1) parents and in 146 F(2) individuals with a female-biased sex ratio (106 females:40 males), regions of seven cDNA loci were analyzed with single-strand conformation polymorphism (SSCP) analysis to identify and orient linkage groups. Genotypes were also scored at 73 random amplified polymorphic DNA (RAPD)-SSCP loci. In addition to the sex locus, at least four QTL affecting sex determination were detected with interval mapping on linkage groups I and II. Alleles at the sex locus cumulatively accounted for approximately 61-77% of the genetic variance in sex. Alleles at QTL adjacent to the sex locus and at a QTL on the opposite end of linkage group I increased the proportion of females, but alleles at a QTL on linkage group I and a second QTL on linkage group II increased the proportion of males. The female-biased sex ratio observed in laboratory colonies of O. triseriatus is most easily explained by the existence of multiple female biased distorter loci, as have been observed in other Diptera.

Amplifying whole insect genomes with multiple displacement amplification.

Many applications in insect genetics require repeated analyses on individual organisms. These include population genetic and genomics, linkage mapping, molecular systematics and map-based positional cloning. However, using the polymerase chain reaction, a limited number of analyses are possible with DNA isolated from whole bodies or parts of small or preserved specimens. We describe the optimization of a new technique, Multiple Displacement Amplification, for 100-400-fold amplification of whole mosquito genomes. We demonstrate that MDA amplifies genomic DNA directly from an adult leg or from organisms as small as a first instar mosquito larva. Genetic polymorphisms revealed at individual loci are the same whether using the original genomic DNA or MDA DNA as a template.

Molecular characterization of California serogroup viruses isolated in Russia.

Nucleotide sequencing was used to characterize unidentified California (CAL) serogroup virus isolates from Russia. These viruses were isolated from mosquitoes and humans during epidemiologic investigations on the role of CAL serogroup viruses in the increased incidence of arboviral encephalitis in Russia. Most of the isolates were identified serologically as snowshoe hare (SSH), Inkoo (INK), and Tahyna (TAH) viruses, but some of the isolates were difficult to classify serologically, suggesting that they could be reassortant viruses. There is evidence that at least 2 of these viruses are not reassortant viruses. Sequence analysis revealed that the Russian viruses differ from other Eurasian and North American CAL serogroup viruses in all of the segments analyzed. They are most closely related to SSH virus. Whether they differ sufficiently to be considered a new group of SSH-like viruses remains to be determined.

Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes.

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.

Novel rearrangements of arthropod mitochondrial DNA detected with long-PCR: applications to arthropod phylogeny and evolution.

Rearrangements of mitochondrial DNA gene order have been suggested as a tool for defining the pattern of evolutionary divergence in arthropod taxa. We have employed a combination of highly conserved insect-based polymerase chain reaction (PCR) primers with long-PCR to survey 14 noninsect arthropods for mitochondrial gene rearrangements. The size of the amplified fragments was used to order the primer containing genes. Five chelicerates exhibit amplicons that are consistent with the presumptive ancestral arthropod mtDNA gene order. These five species comprise two soft ticks, two prostriate hard ticks, and an opilionid. Six other chelicerates, all metastriate hard ticks, have a different arrangement that was originally discovered by this procedure and has been previously detailed in a complete mtDNA sequence. Three new arthropod mtDNA gene arrangements are described here. They were discovered in a terrestrial crustacean (Isopoda) and two myriapods (Chilopoda, centipede; Diplopoda, millipede). These rearrangements include major realignments of some of the large coding regions and two possible new positions for the tRNA(Met) (M) gene in arthropods. The long-PCR approach affords an opportunity to quickly screen divergent taxa for major rearrangements. Taxa exhibiting rearrangements can be targeted for DNA sequencing of gene boundaries to establish the details of the mtDNA organization.

CT screening for lung cancer: not ready for routine practice.

Lung cancer continues to be a major worldwide health problem. Multiple strategies are being explored in an attempt to reduce lung cancer mortality, including a renewed interest in screening. Multiple low-dose spiral computed tomography (CT) trials have been proposed, as proponents predict that small nodules will represent early-stage disease and detecting them will ultimately translate into improvements in outcomes. At this time, however, only prevalence-screening data are available, and it remains to be seen if CT will truly reduce mortality. The appropriate hypothesis-driven studies still must be performed and the results carefully analyzed before CT screening for lung cancer can be accepted as the standard of care.

SSCP analysis of cDNA markers provides a dense linkage map of the Aedes aegypti genome.

An intensive linkage map of the yellow fever mosquito, Aedes aegypti, was constructed using single-strand conformation polymorphism (SSCP) analysis of cDNA markers to identify single nucleotide polymorphisms (SNPs). A total of 94 A. aegypti cDNAs were downloaded from GenBank and primers were designed to amplify fragments <500 bp in size. These primer pairs amplified 94 loci, 57 (61%) of which segregated in a single F(1) intercross family among 83 F(2) progeny. This allowed us to produce a dense linkage map of one marker every 2 cM distributed over a total length of 134 cM. Many A. aegypti cDNAs were highly similar to genes in the Drosophila melanogaster genome project. Comparative linkage analysis revealed areas of synteny between the two species. SNP polymorphisms are abundant in A. aegypti genes and should prove useful in both population genetics and mapping studies.