Rapid Assays for Identification of Members of the Culex (Culex) Pipiens Complex, Their Hybrids, and Other Sibling Species (Diptera: Culicidae)

Smith, Julie L.; Fonseca, Dina M.

doi:10.4269/ajtmh.2004.70.339

Cited by 288 publications

(327 citation statements)

References 27 publications

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“…One hundred larvae were tested at each concentration point. Given that these were wild caught Culex from mixed species collections it was necessary to identify the larvae using a combination of morphological keys (Hopkins, 1952) and molecular assays (Smith and Fonseca, 2004).…”

Section: Association Of Kdr or The Cure1 Insertion Upstream Of Cyp9m1mentioning

confidence: 99%

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Wilding

Smith

Lynd

et al. 2012

Insect Biochemistry and Molecular Biology

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Although cytochrome P450 (CYP450) enzymes are frequently up-regulated in mosquitoes resistant to insecticides, no regulatory motifs driving these expression differences with relevance to wild populations have been identified. Transposable elements (TEs) are often enriched upstream of those CYP450s involved in insecticide resistance, leading to the assumption that they contribute regulatory motifs that directly underlie the resistance phenotype. A partial CuRE1 (Culex Repetitive Element 1) transposable element is found directly upstream of CYP9M10, a cytochrome P450 implicated previously in larval resistance to permethrin in the ISOP450 strain of Cx. quinquefasciatus, but is absent from the equivalent genomic region of a susceptible strain. Via expression of CYP9M10 in E.coli we have now demonstrated time-and NADPH-dependant permethrin metabolism, prerequisites for confirmation of a role in metabolic resistance, and through qPCR shown that CYP9M10 is >20-fold over-expressed in ISOP450 compared to a susceptible strain. In a fluorescent reporter assay the region upstream of CYP9M10 from ISOP450 drove 10x expression compared to the equivalent region (lacking CuRE1) from the susceptible strain. Close correspondence with the gene expression fold-change implicates the upstream region including CuRE1 as a cis-regulatory element involved in resistance. Only a single CuRE1 bearing allele, identical to the CuRE1 bearing allele in the resistant strain, is found throughout Sub-Saharan Africa, in contrast to the diversity encountered in non-CuRE1 alleles. This suggests a single origin and subsequent spread due to selective advantage. CuRE1 is detectable using a simple diagnostic. When applied to Cx. quinquefasciatus larvae from Ghana we have demonstrated a significant association with permethrin resistance in multiple field sites (mean Odds Ratio = 3.86) suggesting this marker has relevance to natural populations of vector mosquitoes. However, when CuRE1 was excised from the allele used in the reporter assay through fusion PCR, expression was unaffected, indicating that the TE has no direct role in resistance and hence that CuRE1 is acting only as a marker of an as yet unidentified regulatory motif in the association analysis. This suggests that a re-evaluation of the assumption that TEs contribute regulatory motifs involved in gene expression may be necessary. 3 IntroductionThe control of a number of pernicious mosquito-borne diseases such as malaria, lymphatic filariasis Chanda et al., 2011;Himeidan et al., 2011; Mathias et al., 2011; Yewhalaw et al., 2011). Given that many resistance mutations are thought to be recessive in expression, development of DNA diagnostics enables disease and agricultural control programmes to identify resistance mechanisms when they are at low frequency and take ameliorating action before they cause economic or health impacts (Hemingway, 2009;Kelly-Hope et al., 2008). As shown in a number of recent studies, resistance alleles can rise towards fixation very rapidly (García et al., 2009;L...

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Section: Association Of Kdr or The Cure1 Insertion Upstream Of Cyp9m1mentioning

confidence: 99%

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Wilding

Smith

Lynd

et al. 2012

Insect Biochemistry and Molecular Biology

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“…pipiens complex and to characterize populations and zones of hybridization (Bourguet et al 1998, Aspen & Savage 2003, Smith & Fonseca 2004, Savage et al 2007, Sanogo et al 2008, Kothera et al 2009). The fragment of the ace-2 locus was amplified from each DNA sample by PCR in a final reaction volume of 50 μL with 2 mM Mg ++ , 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.5 mM of each primer, 0.2 mM dNTP mix, 2 U of Taq DNA polymerase (Invitrogen) and approximately 6.0 ng of genomic DNA.…”

Section: Methodsmentioning

confidence: 99%

Genetic-morphometric variation in Culex quinquefasciatus from Brazil and La Plata, Argentina

Morais

Moratore

Suesdek

et al. 2010

Mem. Inst. Oswaldo Cruz

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Variation among natural populations of Culex (Culex) Key words: Culex pipiens complex -Culex quinquefasciatus -clines -molecular identification -wing morphologyThe Culex (Culex) pipiens complex in the Americas is composed of two main species, Culex (Culex) quinquefasciatus Say, which is adapted to tropical zones and Cx. pipiens L., which is found in temperate zones. In intermediate areas, these two members can mate yielding hybrids and intermediate forms (Barr 1957), through the processes of gene flow and introgression (Humeres et al. 1998, McAbee et al. 2008, Kothera et al. 2009). Mosquitoes of the Cx. pipiens complex are potential vectors of filarid worms in tropical and subtropical areas (Bockarie et al. 2009) and diverse arboviruses, including West Nile virus (Apperson et al. 2004, Cook et al. 2006.Adult females and immature stages of the Cx. pipiens complex are morphologically similar. The differentiation of adult males can be made by analysis of genitalia, which includes measuring the DV/D ratio of the dorsal arms of the aedeagus in the phallosome, as reported by Sundararaman (1949). Because members of the Cx. pipiens complex have a broad geographical distribution, other phenotypical differences beyond those of the male genitalia can be found. For example, some wing characters can also been used for morphologic identification of mosquito species.Financial support: FAPESP (05/50225-2, 06/02622-5) SAM and CM contributed equally to this work (FAPESP 06/57272-9 and 07/1665-5, respectively The taxonomic keys compiled by Forattini (2002) compared wing morphology of adult females. In that report, the subcosta vein (Sc) was reported to bind with the costa vein (C) before the bifurcation of the radial 2+3 vein (R 2+3 ) in Cx. quinquefasciatus, whereas in Cx. pipiens, the Sc vein binds to vein C at the same point or beyond the R 2+3 bifurcation. To our knowledge, these findings have not been investigated in other geographically distant populations. Linam and Nielsen (1962) reported that Bekku (1956), in Northern Japan, also attempted to use wing characteristics [the length of radial cell R 2 (second marginal cell) divided by the length of vein R 3 ] to distinguish adults of the pipiens group. The results of Bekku (1956) were inconclusive, which was later explained when Kamura (1958) found that these particular wing measurements varied with the temperature of the environment. Nielsen and Rees (1961) and Linam and Nielsen (1962) reported that the most reliable method of separating females of Cx. pipiens and Cx. quinquefasciatus was by a wing measurement value obtained by dividing the length of the cell R 2 by the length of the vein R 2+3 (R 2 /R 2+3 ratio). These authors reported the value of this ratio to be about 5.0 in Cx. pipiens and 3.0 or less in Cx. quinquefasciatus, with intermediate values present in the hybrids. However, these studies did not evaluate morphometric data among mosquito populations and climatic/geographic gradients.In insects, the phenotypic variations among members of species complexes have...

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“…pipiens complex. The Crabtree et al (1997), Aspen and Savage (2003), and Smith and Fonseca (2004) PCR diagnostics also were used to differentially identify Cx. p. pipiens and Cx.…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Genetic Differences BetweenCulex pipiensf. molestus andCulex pipiens pipiens(Diptera: Culicidae) in New York

Kent¹,

Harrington²,

Norris³

2007

J Med Entomol

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The definition and phylogenetic placement of the autogenous molestus form of Culex pipiens has puzzled entomologists for decades. We identified genetic differences between Cx. p. pipiens (L.) and Cx. pipiens f. molestus Forskål in the SH60 fragment described previously. Single-strand conformation polymorphism analysis, cloning, and sequencing of this fragment demonstrated high polymorphism within and among individual Cx. p. pipiens, with common SH60 variants shared among individuals from distant locations. In contrast, Cx. pipiens f. molestus from New York City each contained a single SH60 variant, which was not identified in any other Cx. p. pipiens specimens analyzed. Supporting microsatellite analysis demonstrated significant but reduced gene flow between Cx. p. pipiens and Cx. pipiens f. molestus in New York relative to Cx. p. pipiens populations in New York and California. Results are discussed in the context of two contrasting hypotheses regarding the origin of Cx. pipiens f. molestus populations. KeywordsCulex pipiens pipiens; Culex pipiens f. molestus; genetics; microsatellites; West Nile virus Culex pipiens f. molestus Forskål is a morphologically identical ecological biotype of Culex pipiens pipiens (L.) defined by a host of behavioral and physiological characteristics. In contrast to Cx. p. pipiens, Culex pipiens f. molestus has the ability to produce eggs without a vertebrate bloodmeal (autogeny); can mate in confined spaces (stenogamy); foregoes winter diapause; occupies subterranean environments with limited surface access; and feeds readily on mammals, including humans (Mattingly 1952, Vinogradova 2000. The potential public health importance of the mammal-feeding molestus biotype of Cx. p. pipiens as a human disease vector has led to a search for markers to readily identify these two forms.Despite the numerous biological differences that distinguish Cx. pipiens f. molestus from Cx. p. pipiens, reliable identification of Cx. pipiens f. molestus by using morphology and/or molecular tools and phylogenetic placement of this form within the Cx. pipiens complex NIH Public Access Polymerase Chain ReactionThe relative quality of all DNA extractions was evaluated by PCR amplification of a fragment of the mitochondrial NADH dehy-drogenase subunit four (ND4) by using arthropod-specific primers (Simon et al. 1994, Kent andNorris 2005). Molecular identification for all samples was obtained using the Crabtree et al. bromide-stained 2% agarose gels. All gels were run with GeneRuler 100-bp molecular mass marker (MBI Fermentas, Hanover, MD). p. pipiens were purified with the QIAquick PCR Purification or Gel Extraction kits (QIAGEN, Valencia, CA) and cloned using chemically competent TOP10 One Shot Escherichia coli (TOPO TA Cloning kit with pCR 2.1-TOPO vector catalog K4500-01, Invitrogen, Carlsbad, CA) to evaluate differences between SH60 variants. Transformed E. coli were grown at 37°C overnight on LB agar plates containing 50 µg/ml carbenicillin, and transformed colonies were identified by blue-white...

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Rapid Assays for Identification of Members of the Culex (Culex) Pipiens Complex, Their Hybrids, and Other Sibling Species (Diptera: Culicidae)

Cited by 288 publications

References 27 publications

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Genetic-morphometric variation in Culex quinquefasciatus from Brazil and La Plata, Argentina

Genetic Differences BetweenCulex pipiensf. molestus andCulex pipiens pipiens(Diptera: Culicidae) in New York

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