Increased production of mitochondrial reactive oxygen species and reduced adult life span in an insecticide-resistant strain of Anopheles gambiae.

Control of the malaria vector An. gambiae is still largely obtained through chemical intervention using pyrethroids, such as permethrin. However, strains of An. gambiae that are resistant to the toxic effects of pyrethroids have become widespread in several endemic areas over the last decade. The objective of this study was to assess differences in five life-history traits (larval developmental time and the body weight, fecundity, hatch rate, and longevity of adult females) and energy metabolism between a strain of An. gambiae that is resistant to permethrin (RSP), due to knockdown resistance and enhanced metabolic detoxification, and a permethrin susceptible strain reared under laboratory conditions. We also quantified the expression levels of five antioxidant enzyme genes: GSTe3, CAT, GPXH1, SOD1, and SOD2. We found that the RSP strain had a longer developmental time than the susceptible strain. Additionally, RSP adult females had higher wet body weight and increased water and glycogen levels. Compared to permethrin susceptible females, RSP females displayed reduced metabolic rate and mitochondrial coupling efficiency and higher mitochondrial ROS production. Furthermore, despite higher levels of GSTe3 and CAT transcripts, RSP females had a shorter adult life span than susceptible females. Collectively, these results suggest that permethrin resistance alleles might affect energy metabolism, oxidative stress, and adult survival of An. gambiae. However, because the strains used in this study differ in their genetic backgrounds, the results need to be interpreted with caution and replicated in other strains to have significant implications for malaria transmission and vector control.

Disruption of the Wolbachia surface protein gene wspB by a transposable element in mosquitoes of the Culex pipiens complex (Diptera, Culicidae).

Culex pipiens quinquefasciatus Say and Culex pipiens pipiens Linnaeus are sibling species incriminated as important vectors of emerging and re-emerging infectious diseases worldwide. The two forms differ little morphologically and are differentiated mainly based upon ecological, behavioural, physiological and genetic traits. Within the North American zone of sympatry, populations of Cx. p. quinquefasciatus and Cx. p. pipiens undergo extensive introgression and hybrid forms have been reported in nature. Both Cx. p. quinquefasciatus and Cx. p. pipiens are infected with the endosymbiotic bacteria Wolbachia pipientis. Here, we report the presence of a transposable element belonging to the IS256 family (IS256wPip) associated with Wolbachia in both Cx. p. quinquefasciatus and Cx. p. pipiens populations. Using reverse transcriptase PCR and sequence analysis, we show that IS256wPip has disrupted the wspB locus, a paralogue of the Wolbachia outer membrane protein (wspA) gene. The inactivation of the wspB appears to be specific to Cx. p. quinquefasciatus and to hybrids of the two forms, and was not observed in the surveyed Cx. p. pipiens mosquitoes. Our results support the hypothesis of a different origin of North American Cx. p. quinquefasciatus and Cx. p. pipiens populations. The flux of mobile genetic elements in the Wolbachia wPip genome could explain the high level of crossing types observed among different Culex populations. The insertion of IS256wPip into wspB may comprise a genetic candidate for discriminating Wolbachia symbionts in Culex.

Assessment of arbovirus vector infection rates using variable size pooling.

Pool testing of vector samples for arboviruses is widely used in surveillance programmes. The proportion of infected mosquitoes (Diptera: Culicidae) is often estimated from the minimum infection rate (MIR), based on the assumption of only one infected mosquito per positive pool. This assumption becomes problematic when pool size is large and/or infection rate is high. By relaxing this constraint, maximum likelihood estimation (MLE) is more useful for a wide range of infection levels that may be encountered in the field. We demonstrate the difference between these two estimation approaches using West Nile virus (WNV) surveillance data from vectors collected by gravid traps in Chicago during 2002. MLE of infection rates of Culex mosquitoes was as high as 60 per 1000 at the peak of transmission in August, whereas MIR was less than 30 per 1000. More importantly, we demonstrate roles of various pooling strategies for better estimation of infection rates based on simulation studies with hypothetical mosquito samples of 18 pools. Variable size pooling (with a serial pool sizes of 5, 10, 20, 30, 40 and 50 individuals) performed consistently better than a constant size pooling of 50 individuals. We conclude that variable pool size coupled with MLE is critical for accurate estimates of mosquito infection rates in WNV epidemic seasons.

Efficacy of Bacillus thuringiensis israelensis, Bacillus sphaericus and temephos for managing Anopheles larvae in Eritrea.

We evaluated the larvicidal activity of the granular formulation of Bacillus thuringiensis israelensis (Bti) serotype H-14 (Vectobac G, 200 ITU/mg) and Bacillus sphaericus (Bsph) serotype H5a5b (Vectolex CG, 670 Bs ITU/mg) against Anopheles arabiensis and other mosquitoes in breeding habitats in 3 sites, Gash-Barka, Anseba, and Debub zones, in Eritrea. The primary objective was to determine the optimal application rate and duration of effect for Bti and Bsph in representative larval habitats as compared with the organophosphate temephos. The biolarvicides were tested at 100% (high) and 50% (low) of the maximum recommended application rate. Temephos was applied at a rate of 100 ml/ha. At least 4 replicate experiments with Vectobac G (5.6 and 11.2 kg/ha), Vectolex CG (11.2 and 22.4 kg/ha) were conducted in each study site. All 3 larvicides caused significant mortality of the main malaria vector species, An. arabiensis, and other mosquito species (Anopheles cinereus, Anopheles pretoriensis, Culex quinquefasciatus). The larvicidal activity for Bti and Bsph was variable depending upon breeding habitat, mosquito species, and general ecology of the area. Both biopesticides had a similar duration of activity (2-3 wk) and were generally as effective as temephos for these time periods. In some cases, the high and low application rates for Bti and Bsph produced equivalent control over 2-3 wk. The 2 Bacillus biopesticides were less effective in habitats with high algal content and in fast flowing streams primarily because of the inability to penetrate algal mats and dilution effect, respectively. The results show that application of the 2 biolarvicides bimonthly to streambed pools, rain pools, and similar habitats would maintain control of the anopheline mosquito population.

Recovery of commercially produced Bacillus thuringiensis var. israelensis and Bacillus sphaericus from tires and prevalence of bacilli in artificial and natural containers.

We conducted surveys to identify the species of spore-forming bacteria present in natural and artificial containers. Most of our samples came from Illinois. Identification was based on the cellular fatty acid composition of the bacterial cell wall. In addition, we utilized a custom database for commercially produced strains of Bacillus thuringiensis var. israelensis (Bti) and B. sphaericus, to differentiate between larvicidal isolates with commercial or native origin. Native Bti was present at low levels in almost all habitats but was not recovered from bromeliads and metal containers. In temporary woodland pools, 27.9% of the colonies recovered were native Bti. We did not recover larvicidal B. sphaericus in untreated habitats. VectoBac and VectoLex were applied to tires containing water and the tires were sampled 3 months and 9 months after treatment. Isolates of Bti and B. sphaericus with commercial origin were recovered as long as 9 months after application. We noticed numerous cadavers of Aedes triseriatus in several tires 9 months after treatment with VectoBac. We could not determine if this mortality resulted from recycling of Bti in these tires or whether insecticidal crystal proteins from the original treatment were resuspended. Bacillus thuringiensis var. israelensis isolates with commercial ancestry were recovered from untreated tires 9 months after application. Isolates of larvicidal B. sphaericus that differed from the bacteria in VectoLex were also recovered from untreated tires.

Effects of magnetic fields on mosquitoes.

Phylogenetically diverse organisms, including some insects, are able to detect and respond to magnetic fields comparable to the Earth's magnetic field. Because of their tremendous importance to public health, mosquitoes were tested for the presence of remanent ferromagnetic material indicative of a biological compass and also tested for behavioral responses to magnetic fields. Using a superconducting quantum interferometry device, we found that significant remnant was probably due to attraction of ferromagnetic dust onto the surface of live or dead mosquitoes. Most mosquitoes placed in a 1.0-gauss, uniform magnetic field moved until they were oriented parallel to the field. Two of 3 species of mosquitoes tested took fewer blood meals in a rotating magnetic field than in the Earth's normal magnetic field.

Field efficacy of commercial antimosquito products in Illinois.

We evaluated the efficacy of commercial antimosquito products in field trials in Illinois in June 1998 by comparing mosquito landing rates. Products tested were a sonic mosquito repeller, an insect killing grid using ultraviolet light and 1-octen-3-ol as lures, mosquito smoke coils containing a pyrethroid, citronella candles, the mosquito plant Pelargonium citrosum, and a N,N-diethyl-3-methylbenzamide (deet)-impregnated wrist band. The sonic mosquito repeller, insect killing grid, and mosquito smoke coils were evaluated in 16 trials over 5 days; the citronella candles and mosquito plants in II trials over 4 days; and the wrist bands in 4 trials on 1 day. In all 3 studies, we compared landing rates with the antimosquito products to both positive (topical application of a deet formulation) and negative (no treatment) controls. The deet topical repellent had a consistently lower landing rate than all the nontopically applied products tested. However, the mosquito coils and the deet-impregnated wrist bands did significantly reduce mosquito landing rates relative to untreated controls.

Differentiation of Aedes triseriatus (Say) from Aedes hendersoni Cockerell (Diptera: Culicidae) by restriction fragment length polymorphisms of amplified ribosomal DNA.

Aedes triseriatus is the primary vector of LaCrosse (LAC) virus, which can cause encephalitis, especially in young children. Aedes hendersoni, a sibling species of Ae. triseriatus, has a salivary gland barrier to LAC virus and, therefore, is not considered a vector of this virus. Adults of Ae. triseriatus are morphologically indistinguishable from those of Ae. hendersoni, and the two species are sympatric in the eastern United States. A definitive method of identifying field specimens is an important part of any disease surveillance program, particularly in the case of LAC virus. This study identifies restriction enzymes that produce species-specific restriction fragment length polymorphisms (RFLPs) from amplified ribosomal (r) DNA. In addition, sequences of the internal transcribed spacers 1 and 2 and the 5.8S regions of the rDNA were used to confirm the RFLP patterns. This study is the first to compare nucleotide sequences from Ae. triseriatus and Ae. hendersoni.

Evaluation of methylated soy oil and water-based formulations of Bacillus thuringiensis var. Israelensis and Golden Bear Oil (GB-1111) against anopheles quadrimaculatus larvae in small rice plots.

The efficacy of formulations containing methylated soybean oil (MSO) alone and with technical-grade Bacillus thuringiensis var. israelensis (Bti) were compared to Golden Bear Oil (GB-1111) and a water-based Bti formulation against 3rd- to 4th-stage Anopheles quadrimaculatus larvae confined to sentinel cages in small rice plots. Three replicates each of MSO with 2% Pyroter added as a surfactant (MSO + PYR), MSO with 2% Pyroter and 4 g of Bti technical powder (MSO + PYR + Bti), GB-1111, a water-based formulation with 4 g of Bti technical powder (Bti + water), and untreated controls were performed. Mosquito larvae were introduced on the 1st day of treatment and at 4 days posttreatment. Mortality was recorded at 24 and 48 h posttreatment for the 1st installation and at 5 days posttreatment for the 2nd installation. The Bti + water formulation provided 71% control and the MSO + PYR + Bti formulation achieved 64% control, whereas MSO + PYR and GB-1111 produced 16 and 18% control, respectively, at 24 h posttreatment. With the exception of MSO + PYR + Bti, which decreased by 2%, the mean percent control increased slightly at 48h posttreatment across remaining treatments, with Bti + water obtaining 72% control. This was significantly higher than GB-1111, which achieved 23% control at 48 h posttreatment. The MSO + PYR and MSO + PYR + Bti formulations yielded 56 and 62% control, respectively, during the same interval and were not significantly different from one another. Formulations containing MSO + PYR exhibited delayed activity similar to GB-1111, with all formulations except MSO + PYR + Bti providing greatest control at 48 h posttreatment. Both MSO formulations (MSO + PYR + Bti and MSO + PYR) were statistically comparable to Bti + water and GB-1111, respectively, at 24 and 48 h posttreatment. None of the formulations exhibited a residual activity adequate enough to control An. quadrimaculatus larvae for up to 5 days.

Duration of activity of the microbial larvicide VectoLex CG (Bacillus sphaericus) in Illinois catch basins and waste tires.

The duration of activity of a formulation of Bacillus sphaericus, VectoLex CG, for control of Culex species was evaluated in 338 catch basins in Urbana, IL, and compared to Altosid in 346 catch basins in Champaign, IL. The activity of VectoLex in car and truck waste tires was evaluated in a tire dump located in Pembroke Township, IL. In catch basins, 1 g of VectoLex per catch basin gave the same control as one Altosid briquet. Both larvicides were effective against Culex sp. in catch basins for 1 month, and the duration of control with VectoLex lasted 44 days in one catch basin. VectoLex was considerably cheaper to apply than Altosid briquets, at 0.64 cents per catch basin compared to 90.75 cents, respectively. However, the Altosid briquets were judged to be easier to apply from a vehicle than VectoLex granules. VectoLex (22.6 kg) was used to treat approximately 6,000 car and truck tires; some of the tires were in direct sunlight whereas others were shaded. Aedes triseriatus was the dominant species in these tires. Tires treated with VectoLex contained significantly fewer mosquitoes than control tires, and even 65 days after application, control tires were 16.7 times more likely to contain larvae. We conclude that VectoLex was effective when used in Illinois catch basins and tire dumps, and emphasize that it is more appropriate to base tire treatment rates on the total number of tires present than on a kilogram per hectare basis.

Culiseta impatiens (Walker) in Illinois, a new state record.

We report the 1st collection of Culiseta impatiens (Walker) from Illinois. Larvae of Culiseta subsequently identified as Cs. impatiens were collected from roadside ditches near Savoy, Champaign County, in east central Illinois in June 1997. This extends the known distribution of this species further south and east in the midwestern United States.

Natural habitats of Aedes Aegypti in the Caribbean--a review.

Natural breeding habitats of Aedes aegypti in the Caribbean region were reviewed by conducting larval surveys in Trinidad. Puerto Rico, and the U.S. Virgin Islands and referring to records from the Mosquitoes of Middle America project. Twelve types of natural habitats were recorded: rock holes (9.7%), calabashes (2.4%), tree holes (19.5%), leaf axils (4.8%), bamboo joints (14.9%), papaya stumps (7.3%), coconut shells (4.8%), bromeliads (7.3%), ground pools (14.9%), coral rock holes (9.7%), crab holes (2.4%), and conch shells (7.3%), of which the coconut shell and calabash habitats were new to the Caribbean. The countries having the highest prevalence of natural habitats were Trinidad. Puerto Rico, and Jamaica, with 9 types (22.0%), 7 types (17.0%), and 6 types (14.6%), respectively. The distribution of natural habitats of Ae. aegypti in the Caribbean region is discussed in relation to vector control measures.

Comparison of the cellular fatty acid composition of a bacterium isolated from a human and alleged to be Bacillus sphaericus with that of Bacillus sphaericus isolated from a mosquito larvicide.

The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography. The CFA composition of B. sphaericus 2362, isolated from a microbial larvicide, and those of B. sphaericus reference strains obtained from public collections were also determined. Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages. Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain. NCTC 11025 and the type strain of B. sphaericus, ATCC 14577, were mixed; all other isolates were monotypic. The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid. NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify. We could distinguish among six DNA homology groups of B. sphaericus by using fatty acids. Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b. We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B. sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.

Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.

Species-level identification of Acanthamoeba isolates is difficult and gives little or no indication of the isolate's pathogenicity. We identified two amplification-based genetic markers that were highly correlated with pathogenicity in Acanthamoeba spp. One marker, designed to amplify a 485-bp fragment of the small-subunit ribosomal RNA gene (ssrDNA), was preferentially amplified from the nonpathogenic strains; amplifications from the pathogenic strains yielded anomalous fragments of 650 and 900 bp. A second marker was developed on the basis of the anomalous 650-bp fragment. Primers to this sequence preferentially amplified a noncoding locus (called Ac6) only from the pathogenic strains. These two genetic markers may be useful for identification of pathogenic Acanthamoeba spp. strains.

Field trials of VectoLex CG, a Bacillus sphaericus larvicide, in Illinois waste tires and catch basins.

The susceptibilities of Aedes triseriatus (Say), Anopheles punctipennis (Say), Culex restuans (Theobald), and Culex pipiens (L.) larvae to VectoLex CG were determined. VectoLex, formulated on corncob granules (10-14 mesh) was applied to 2 tire dumps and numerous catch basins located in east-central Illinois. VectoLex, formulated as effervescent tablets, was also applied to catch basins. In a sunlit dump, there was a 99.6% reduction in Ae. triseriatus, Cx. restuans, and Cx. pipiens larvae as long as 32 days after treatment, and no pupae were recovered during this interval. There was still a 71.6% larval reduction 74 days after treatment. There was an overall reduction of 22% for An. punctipennis larvae. In a shaded dump, no larval Ae. triseriatus, Cx. restuans, or Cx. pipiens were recovered for 25 days after treatment, and no pupae were recovered 25-67 days after treatment. There was still a 68.7% larval reduction 74 days after treatment. An. punctipennis was unaffected. In one catch basin study, VectoLex was comparable to Altosid (average of 46 days vs. 50 days until larvae were recovered from catch basins). As the summer progressed, the duration of VectoLex control was reduced to 30 days. VectoLex effervescent tablets (evaluated mid-August through September) gave 18-day control. VectoLex was effective against Ae. triseriatus, Cx. restuans, and Cx. pipiens, but was not effective against An. punctipennis in waste tires. VectoLex was effective against Cx. restuans and Cx. pipiens in catch basins.

Aedes albopictus and Ae. triseriatus eggs survive waste tire processing.

Tires that were either crudely chopped or more finely processed into shreds contained viable eggs. Field-collected remnants of 2-3 chopped tires contained viable Aedes albopictus eggs. After shredding tires seeded with mosquito eggs, 34 (4.6%) of an estimated 746 Ae. albopictus eggs and 21 (2.7%) of an estimated 774 Aedes triseriatus eggs survived. Chopped and shredded tire remnants may serve as a means of dispersing mosquitoes.