High degree of single nucleotide polymorphisms in California Culex pipiens (Diptera: Culicidae) sensu lato.

Resolution of systematic relationships among members of the Culex pipiens (L.) complex has important implications for public health as well as for studies on the evolution of sibling species. Currently held views contend that in California considerable genetic introgression occurs between Cx. pipiens and Cx. quinquefasciatus Say, and as such, these taxa behave as if they are a single species. Development of high throughput SNP genotyping tools for the analysis of Cx. pipiens complex population structure is therefore desirable. As a first step toward this goal, we sequenced 12 gene fragments from specimens collected in Marin and Fresno counties. On average, we found a higher single nucleotide polymorphism (SNP) density than any other mosquito species reported thus far. Coding regions contained significantly higher GC content (median 54.7%) than noncoding regions (42.4%; Wilcoxon rank sum test, P = 5.29 x 10(-5)). Differences in SNP allele frequencies observed between mosquitoes from Marin and Fresno counties indicated significant genetic divergence and suggest that SNP markers will be useful for future detailed population genetic studies of this group. The high density of SNPs highlights the difficulty in identifying species within the complex and may be associated with the large degree of phenotypic variation observed in this group of mosquitoes.

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.

Ecological and genetic relationships of the Forest-M form among chromosomal and molecular forms of the malaria vector Anopheles gambiae sensu stricto.

BACKGROUND: Anopheles gambiae sensu stricto, one of the principal vectors of malaria, has been divided into two subspecific groups, known as the M and S molecular forms. Recent studies suggest that the M form found in Cameroon is genetically distinct from the M form found in Mali and elsewhere in West Africa, suggesting further subdivision within that form. METHODS: Chromosomal, microsatellite and geographic/ecological evidence are synthesized to identify sources of genetic polymorphism among chromosomal and molecular forms of the malaria vector Anopheles gambiae s.s. RESULTS: Cytogenetically the Forest M form is characterized as carrying the standard chromosome arrangement for six major chromosomal inversions, namely 2La, 2Rj, 2Rb, 2Rc, 2Rd, and 2Ru. Bayesian clustering analysis based on molecular form and chromosome inversion polymorphisms as well as microsatellites describe the Forest M form as a distinct population relative to the West African M form (Mopti-M form) and the S form. The Forest-M form was the most highly diverged of the An. gambiae s.s. groups based on microsatellite markers. The prevalence of the Forest M form was highly correlated with precipitation, suggesting that this form prefers much wetter environments than the Mopti-M form. CONCLUSION: Chromosome inversions, microsatellite allele frequencies and habitat preference all indicate that the Forest M form of An. gambiae is genetically distinct from the other recognized forms within the taxon Anopheles gambiae sensu stricto. Since this study covers limited regions of Cameroon, the possibility of gene flow between the Forest-M form and Mopti-M form cannot be rejected. However, association studies of important phenotypes, such as insecticide resistance and refractoriness against malaria parasites, should take into consideration this complex population structure.

Identification of Culex pipiens complex mosquitoes in a hybrid zone of West Nile virus transmission in Fresno County, California.

Culex pipiens sensu lato mosquitoes were collected from 24 gravid traps (mid-June to mid-October, 2005) in Fresno County, CA. Captured gravid females were allowed to oviposit before sibling species identification by Ace.2 PCR and detection of West Nile virus (WNV) RNA by RT-PCR were performed on the mother and her offspring. Of the 442 Cx. pipiens s.l. female mosquitoes collected, 88 were positive for WNV viral RNA (peaked in August) with no significant differences among complex members or habitat. Vertical transmission was detected in 4 out of 20 families originating from WNV-positive mothers, however, in only a small number of offspring from each family. Out of 101 families that had PCR-based maternal and offspring identifications, the offspring from 15 families produced inexplicable amplicon patterns, suggesting ambiguities in the PCR assay identifications. Male genitalia (DV/D ratio) and Ace.2 PCR identifications revealed numerous discrepancies in our ability to accurately determine the identity of Cx. pipiens complex members in the hybrid zone of Fresno County.

A detailed larval salivary gland polytene chromosome photomap for Culex quinquefasciatus (Diptera: Culicidae) from Johannesburg, South Africa.

The suitability of polytene chromosomes for population genetic and in situ DNA physical mapping was investigated in Johannesburg, South Africa, Culex quinquefasciatus Say and Culex pipiens L.. The most defined and consistent polytene spreads were obtained from salivary glands of early fourth instars from Cx. quinquefasciatus. A photomap is provided for a Cx. quinquefasciatus colony originating from Johannesburg. Variations in banding patterns, chromosome aberrations, and paracentric inversions within the colony and comparisons with previous published descriptions are discussed. Chromosomes of sufficient quality were obtained such that a DNA coding for an odorant binding protein could be mapped by colorimetric in situ hybridization.

Influence of temperature and concentration of VectoBac on control of the salt-marsh mosquito, Ochlerotatus squamiger, in Monterey County, California.

Laboratory susceptibility bioassays were conducted to determine the efficacy of VectoBac TP (Bacillus thuringiensis var. israelensis [Bti]) at different concentrations and temperatures against the salt-marsh mosquito Ochlerotatus squamiger. Bioassays on late 3rd- and early 4th-stage larvae, read at 72 h and 14 degrees C produced an LD90 of 0.223 mg/liter, whereas more than double this dose was required to produce similar mortality at 6 degrees C. A field trial in the winter of 2001-02 of an aerially applied VectoBac TP formulation in Salinas, CA, corroborated laboratory bioassay observations by producing 97-100% control of Oc. squamiger at 72 h postapplication. Inconsistencies in mortality with field applications of VectoBac TP previously observed by North Salinas Valley Mosquito Abatement personnel were most likely caused by uneven application rates and varying temperatures and water volumes.

Pyrethroid tolerance in Culex pipiens pipiens var molestus from Marin County, California.

In May 2001 a sample of Culex pipiens pipiens variety molestus Forskål from Marin County, California, collected as larvae and reared to adults, was found to show reduced resmethrin and permethrin knock-down responses in bottle bioassays relative to a standard susceptible Cx. pipiens quinquefasciatus Say colony (CQ1). Larval susceptibility tests, using CQ1 as standard susceptible, indicated that the Marin mosquitoes had LC50 resistance ratios of 18.3 for permethrin, 12 for deltamethrin and 3.3 for pyrethrum. A colony of Marin was established and rapidly developed higher levels of resistance in a few generations after exposure to permethrin as larvae. These selected larvae were shown to cross-resist to lambda-cyhalothrin as well as to DDT. However, adult knock-down time in the presence of permethrin, resmethrin and pyrethrum was not increased after increase in tolerance to pyrethroids as larvae. Partial and almost complete reversion to susceptibility as larvae was achieved with S, S, S-tributylphosphorotrithioate and piperonyl butoxide (PBO), respectively, suggesting the presence of carboxylesterase and P450 monooxygenase mediated resistance. Insensitive target site resistance (kdr) was also detected in some Marin mosquitoes by use of an existing PCR-based diagnostic assay designed for Cx. p. pipiens L mosquitoes. Carboxylesterase mediated resistance was supported by use of newly synthesized novel pyrethroid-selective substrates in activity assays. Bottle bioassays gave underestimates of the levels of tolerance to pyrethroids of Marin mosquitoes when compared with mortality rates in field trials using registered pyrethroid adulticides with and without PBO. This study represents the first report of resistance to pyrethroids in a feral population of a mosquito species in the USA.

Differences in extent of genetic introgression between sympatric Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in California and South Africa.

Comparisons of five morphological characters, 12 enzyme electrophoresis profiles, and Wolbachia pipientis infection rates were used to characterize populations of members of the Culex pipiens L. complex in California and South Africa. In South Africa, male phallosome DV/D ratio, male maxillary palp index, branching of siphonal seta 1a, the enzyme locus Mdhp-1, and W. pipientis infection rates proved highly diagnostic for separating Culex quinquefasciatus from Cx. pipiens phenotypes. In Johannesburg, where sympatric members of the Cx. pipiens complex were analyzed as one population, a significant Wahlund Effect was observed in the enzyme loci such as Ao, 6-Pgdh, Mdh-2, and Pgm. In California, all populations of the Cx. pipiens complex were in Hardy Weinberg equilibrium at all polymorphic enzyme loci examined. Additionally, in California, all populations had similar W. pipientis infection rates and appeared morphologically identical (except for DV/D ratio, in extreme north and south). These findings indicate that in South Africa, Cx. pipiens and Cx. quinquefasciatus remain as genetically distinct populations and behave as separate species. Conversely, in California, there is considerable genetic introgression between Cx. pipiens and Cx. quinquefasciatus, and they behave as a single species.

High level methoprene resistance in the mosquito Ochlerotatus nigromaculis (Ludlow) in central California.

In the summer of 1998, failures of methoprene field applications to control the mosquito Ochlerotatus nigromaculis (Ludlow) were noticed in several pastures in the outskirts of Fresno, California, USA. Effective control with methoprene had been achieved for over 20 years prior to this discovery. Susceptibility tests indicated that the Fresno Oc nigromaculis populations had developed several thousand-fold higher LC50 and LC90 tolerance levels to methoprene compared with methoprene-naïve populations. The synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate and 3-octylthio-1,1,1-trifluoro-2-propanone had little synergistic effect, suggesting that the mechanism of methoprene tolerance was not mediated by P450 monooxygenase or carboxylesterase enzyme degradation. As part of initiating a resistance management strategy, partial reversion back to methoprene susceptibility was achieved in a resistant population after six consecutive applications of Bacillus thuringiensis israelensis Goldberg & Marga coupled with two oil and two pyrethrum + PBO applications.