Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insect-associated Wolbachia form a monophyletic clade in the α-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.

Methods for the estimation and comparison of survival rates are considered when data arises from a release of individuals followed by a sequence of recaptures, with recaptured individuals removed from the population. It is shown that commonly used methods based on linear regression of the log of recapture numbers versus time can lead to substantial errors if individuals are removed from the population. A general nonlinear regression approach is proposed combined with bootstrap techniques for obtaining confidence intervals and tests of hypotheses. Simulations demonstrate that these techniques perform well using data from an Aedes aegypit L. mark-release-recapture study in Thailand.

Infection rates of Trypanosoma cruzi Chagas (in the blood-sucking bug Dipetalogaster maximus [Uhler]) were determined from specimens collected at 12 localities in the Cape Region of Baja California Sur, Mexico. Eight collection sites were located in the tropical dry forest, two in desert shrub, and two in the ecotone between these two communities. Of the 245 D. maximus collected, 65% were first and second instar nymphs; 32.6% were third, fourth, and fifth instar nymphs; and 2.4% were adults. The highest proportion of specimens came from El Fandango (30%) and San Bartolo (23%) canyons and La Cruz hill (12%) in the mountain slopes facing the Gulf of California. In feces from individual bugs analyzed for T. cruzi, we found an overall infection rate of 7.0%. Infection rates increased from 4.1% in second instars to 42.% in fifth instars. High infection rates were found in bugs collected from La Poza (38.4%), El Gato (27.2%), and El Pedregoso (25%) hills; low infection rates were found in specimens from La Cruz hill and San Bartolo canyon. Specimens from some collection sites were not infected with T. cruzi.

Experiments were conducted to develop an agar-based medium for rearing immature horn flies, Hematobia irritans (L.). Larval survival was determined on sterilized manure inoculated with pure and mixed cultures of Acinetobacter sp., Bacillus pumilus Meyer & Gottheil, Comamonas acidovorans den Dooren de Jong, Pseudomonas mendocina Palleroni, Flavobacterium sp. and Empedobacter breve (Holmes & Owen). Rearing larvae on mixed cultures enhanced pupal weight but not survival. Horn fly larvae failed to survive when reared on standard bacteriological media inoculated with pure and mixed cultures of Acinetobacter sp., P. mendocina, and C. acidovorans. Larvae completed development on a minimal medium supplemented with alfalfa, egg proteins, and vitamins. Medium with low alfalfa content (30 g alfalfa/500 ml minimal medium) had enhanced survival when supplemented with egg yolk protein and vitamins. Medium with high alfalfa content (130 g alfalfa/500 ml minimal medium) had enhanced survival when supplemented with whole egg protein and vitamins. Survival was also favored when media were inoculated with pure cultures of Acinetobacter or Acinetobacter mixed with either Pseudomonas or Comamonas. Individual plates could support larvae developing from up to 40 eggs, and survival was least variable when plates were inoculated with greater numbers of eggs. This rearing system shows promise as a means for conducting standardized bioassays on a meridic diet.

Mitochondrial diversity in house flies was examined by using the single-strand conformation polymorphism method in house flies, Musca domestica L. sampled in six zoogeographical subregions in the New World. The number of haplotypes and haplotype diversities were homogeneous among subregions, but a strong spatial component was found in the distribution of particular haplotypes. Nei’s differentiation index among subregions, G_RT, was 0.53 and that among populations within subregions, G_PR, was 0.31. Greater genetic differentiation was found among populations in the Nearctic than in the Neotropics. Haplotype frequency distributions in two of three Nearctic subregions deviated from that expected under the neutral infinite allele model, suggesting the existence of differential selection patterns.

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.

Esterase activity was present in the integument of adult female Boophilus microplus (Canestrini) ticks that are resistant to organophosphates (OP). Three esterases were purified from adult integument, which hydrolyze the substrates p-nitrophenylacetate and β-naphthyl acetate after comparison of OP-resistant strain and an OP-susceptible strains. The esterases purified by ion-exchange chromatography were characterized using different esterase inhibitors; eserine sulfate, diethyl p-nitrophenyl phosphate (paraoxon), para-hydroxyl-mercuribenzoate (pHMB), and diisopropylphosphofluoridate (DFP). All of the esterases had a molecular mass of 64 Kd (PAGE), but were characterized based on the esterase inhibitor effects as a B-esterase with β-naphthyl acetate affinity, a carboxylesterase with β-naphthyl acetate and p-nitrophenyl acetate affinity, and one A-Esterase (nonspecific esterase) with p-nitrophenyl acetate affinity. The described esterases are an important detoxification mechanism in B. microplus ticks at the integument. We describe also a microplate biochemical assay for the detection of esterase activity in the tick integument, potentially a useful tool to detect esterase-mediated OP resistance in B. microplus ticks.

In an area of India where the main rural malaria vector, Anopheles culicifacies Giles, has developed triple resistance to DDT, HCH, and malathion sprayed indoors in antimalaria program, bifenthrin (10% wettable powder) was evaluated in a randomized house-scale trial between July 1999 and March 2000. Entomological impact of four serial doses of bifenthrin (25, 50, 100, and 200 mg/m²) sprayed in rooms in five villages was compared with malathion (2 g/m²) and unsprayed control. An. culicifacies was 100% susceptible to bifenthrin (0.1%), but only 57% to malathion (5%) test papers. Contact bioassays were carried out on sprayed surfaces for 24 wk, and 24 h mortality in An. culicifacies was recorded. Bifenthrin 100- and 200-mg doses caused ≥80% mortality until 24 wk. The 50-mg dose caused ≥80% mortality on tin, wood, and mud surfaces for 24 wk, and on brick walls for 16 wk. Bifenthrin 25-mg dose produced ≥80% mortality for 24 wk on tin, 20 wk on mud walls, 16 wk on brick walls, and 8 wk on wood surfaces. Persistence of ≥80% mortality did not differ for 25- and 50-mg doses on any surface except on wood (P < 0.05). Malathion sprayed in three rounds of 6 wk apart caused ≥80% mortality for 16 wk on the brick and mud walls, and for 20 wk on the tin and wood surfaces. Bifenthrin 25- and 50-mg doses produced a similar impact on the densities of An. culicifacies and other mosquitoes but a superior one to malathion or control. Bifenthrin 25-mg dose caused least excito-repellency. Overall, efficacy of bifenthrin was superior to malathion. Considering the duration of the persistence of significant insecticidal action of bifenthrin on the most common surfaces (mud and brick walls), least excito-repellency and a relative impact on the mosquito densities, the 25-mg dose was the most superior among all the four doses evaluated.

We developed a rapid and economical in vitro procedure with which to evaluate the efficacy of candidate repellents against chiggers. The procedure requires only 5 min and a small number of chiggers to obtain a valid estimate of the median effective dose. We used this procedure to evaluate the repellent activity of 11 compounds against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. Median effective doses were determined for 10 of the 11 compounds. DM-165-2 (N,N-diethyl-3-flurobenzamide) was the only compound that was significantly more effective than deet.

A new United States (U.S.) self-supporting low-profile bednet was designed by Walter Reed Army Institute of Research in collaboration with Breakthrough Technologies. The bednet incorporated permethrin-impregnated screening into a frame that erected automatically when removed from its bag. The new U.S. bednet was compared with the current Australian Defense Force (ADF) mosquito bednet at Buka Island, North Solomons Province, Papua New Guinea, in March 1999. At the time of the test, Anopheles farauti Laveran was the most abundant biting mosquito. Both bednet types provided >97.8% protection compared with an unprotected collector. The untreated U.S. Army prototype bednet provided better protection than the untreated ADF bednet against mosquitoes entering the bednet during the night.

Larvae of 12 mosquito species were collected from abandoned tire piles at peridomestic and forested sites in Nicholas County, WV, from March through November of 2001. No larvae were found in March, but the numbers of species increased to 10 by July and remained relatively constant, at 9–11 in any given month, throughout November. Larvae of Ochlerotatus triseriatus (Say), the most commonly encountered species in every month of collection, were significantly more likely to be found in forested tire pile sites. Conversely, Culex restuans Theobald, Anopheles punctipennis (Say), Cx. territans Walker, and Aedes albopictus (Skuse) larvae were significantly more likely to be found in peridomestic tire piles. Larvae of the remaining seven species were either found in equal proportions at peridomestic and woodland sites, or there were too few collections to make statistical inferences. Opportunities for competitive interactions between Ae. albopictus and Oc. triseriatus in Nicholas County would be minimized because the peak occurrence of the two species differ temporally and spatially.

Midgut contents of Ornithodoros moubata showed strong antibacterial activity against Staphylococcus aureus. A combination of reversed-phase chromatography and mass spectrometric analysis was used to isolate two antibacterial peptides from the tick midgut lumen. Partial amino acid sequences by Edman degradation of these two peptides showed they are identical with the 1–11 and 3–19 portions of rabbit α hemoglobin. Host rabbit α hemoglobin appears to be cleaved between Met³² and Phe³³ to produce these two antibacterial peptides. Isolation of a host bovine hemoglobin fragment with antimicrobial activity has been demonstrated in the Ixodid tick, Boophilus microplus (Fogaca et al. 1999). Similar immune mechanisms in the two major families of ticks, Ixodidae and Argasidae, appear to use the hemoglobin of the host as an antimicrobial agent in midgut defense.

Australian mosquitoes were evaluated for their ability to become infected with and transmit a Torres Strait strain of Japanese encephalitis virus. Mosquitoes, which were obtained from either laboratory colonies and collected using Centers for Disease Control and Prevention light traps baited with CO₂ and octenol or reared from larvae, were infected by feeding on a blood/sucrose solution containing 10^4.5±0.1 porcine stable-equine kidney (PS-EK) tissue culture infectious dose₅₀/mosquito of the TS3306 virus strain. After 14 d, infection and transmission rates of 100% and 81%, respectively, were obtained for a southeast Queensland strain of Culex annulirostris Skuse, and 93% and 61%, respectively, for a far north Queensland strain. After 13 or more days, infection and transmission rates of >90% and ≥50%, respectively, were obtained for southeast Queensland strains of Culex sitiens Wiedemann and Culex quinquefasciatus Say, and a far north Queensland strain of Culex gelidus Theobald. Although infection rates were >55%, only 17% of Ochlerotatus vigilax (Skuse) and no Cx. quinquefasciatus, collected from far north Queensland, transmitted virus. North Queensland strains of Aedes aegypti L., Ochlerotatus kochi (Dönitz), and Verrallina funerea (Theobald) were relatively refractory to infection. Vertical transmission was not detected among 673 F₁ progeny of Oc. vigilax. Results of the current vector competence study, coupled with high field isolation rates, host feeding patterns and widespread distribution, confirm the status of Cx. annulirostris as the major vector of Japanese encephalitis virus in northern Australia. The relative roles of other species in potential Japanese encephalitis virus transmission cycles in northern Australia are discussed.

The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt, and Brenner was discovered in blacklegged ticks, Ixodes scapularis Say at Rondeau Provincial Park, Ontario, Canada. During this 2-yr study, spirochetes were found in B. burgdorferi-positive I. scapularis larvae attached to B. burgdorferi-infected white-footed mice, Peromyscus leucopus Rafinesque. Isolates of B. burgdorferi were cultured from blacklegged tick adults, and confirmed positive with polymerase chain reaction by targeting OspA and rrf (5S)-rrl (23S) genes. These findings show an endemic area for B. burgdorferi within an established population of I. scapularis at Rondeau Provincial Park.

The recent outbreaks of West Nile (WN) encephalitis and St. Louis encephalitis (SLE) in the United States have highlighted the need for rapid and specific methods of detecting arboviral antigens in mosquitoes. We evaluated rapid, field-usable assays for detecting and differentiating WN and SLE viruses in mosquito pools, based on a patent-pending, immunochromatographic technology (VecTest) formatted on a dipstick. The device provides results in less than 20 min and can be used in laboratories with adequate containment facilities. In laboratory assessments, both the SLE and WN virus tests demonstrated sensitivity comparable with that of an antigen capture ELISA, but less than can be achieved with Vero cell plaque or reverse-transcriptase polymerase chain reaction assays. There was no evidence of cross-reaction when tested with high concentrations of heterologous flavivirus antigens or with Eastern equine encephalitis or Western equine encephalitis viruses. Both the WN and SLE dipstick tests delivered a clear positive result with a single positive specimen in a pool of 50 mosquitoes. This virus assay technology reduces the time required to obtain test results and will allow rapid medical threat assessment and effective targeting of vector control measures.

Genetic sequences characteristic of Borrelia lonestari (Barbour et al. 1996) were detected in two pools of adult Amblyomma americanum (L.) from Tennessee, corresponding to an estimated minimum field infection rate of 8.4 infected ticks/1000 adults. DNA amplification was conducted using primers derived from the B. lonestari flagellin gene that would also amplify Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt, and Brenner). Species-specific, internal probes were then used to differentiate between genetic sequences of the spirochetes. Subsequent nucleotide sequencing confirmed the presence of B. lonestari in A. americanum; B. burgdorferi was not detected. This represents the first report of B. lonestari from Tennessee, and suggests that Lyme-like illness may occur in Tennessee.

Hemaphysalis leporispalustris (Packard), Ixodes brunneus Koch, Ixodes cookei Packard, Ixodes dentatus Marx, and Ixodes texanus Banks were collected during a 3-yr study of pathogen–tick–host interactions in southeastern Missouri. H. leporispalustris was collected from the eastern cottontail rabbit, Northern bobwhite, and Carolina wren, and it was active all year. I. brunneus was collected by drag and from passerine birds during December, March, and April. I. cookei was collected from raccoons and mink during April, June, September, October, and November. I. dentatus was collected from the cottontail rabbit and Carolina wren throughout the year. I. texanus was collected from the eastern gray squirrel, Virginia opossum, and raccoon throughout the year.

In May 2000, a dead porcupine (Erethizon dorsatum Culiver) was found on an infrequently traveled dirt road at Camp Ripley, MN. The presence of late instar Calliphoridae suggested that the porcupine died within the past 4 to 7 d. Adult carrion (Silphidae) and rove (Staphylinidae) beetles were observed under the carcass. In June, a large number of adult American dog ticks, Dermacentor variabilis (Say), were observed questing on the porcupine and the surrounding grass. Six zones were established around the carcass, and each zone was sampled for ticks once a month from June through September. Ticks were captured in June and July, and 93% were captured within 2 m of the carcass. Gases released as part of the decomposition process were believed to attract the ticks to the carcass.

Anopheles quadriannulatus Theobald historically has been reported from southern Africa, Zanzibar islands, and Ethiopia. However, based on evidences of genetic incompatibility between crosses of South African and Ethiopian populations, the population from Ethiopia was recently reported as a distinct species designated as An. quadriannulatus sp. B. An. quadriannulatus sp. A, denoted the southern African population. To distinguish the two populations, the IGS (intergenic spacer) region of rDNA was sequenced to design a primer specific for An. quadriannulatus sp. B. A cocktail polymerase chain reaction (PCR) involving Anopheles gambiae Giles universal (UN) primer, the new primer and other primers specific for members of the An. gambiae complex produced the expected diagnostic products for the respective species. Using extracted DNA and crushed body parts as sources of template DNA, this assay was reliably used to identify samples of An. quadriannulatus sp. B.

Ixodes schulzei Aragão and Fonseca was described from Brazil in 1951 based on female ticks collected on wild rats from the states of Rio de Janeiro and Santa Catarina. Since that time, there have been no additional reports of I. schulzei in the literature. We report two new records of I. schulzei: a female collected on the water rat Nectomys squamipes (Brants) from Minas Gerais State, and another female from this same host species from São Paulo State. This last specimen was engorged and oviposited fertile eggs in the laboratory. Larvae hatched from these eggs were used for subsequent infestations under laboratory conditions, as were nymphs obtained from the engorged larvae. Naive laboratory rats (Rattus norvegicus Berkenhout) and wild mice (Calomys callosus Rengger) were used to feed ticks. C. callosus was a more suitable host than R. norvegicus, as significantly more ticks (P < 0.05) were recovered and successfully molted after feeding on the former host species. A significantly (P < 0.05) greater proportion of larvae detached from C. callosus during daylight (71.3%) when compared with those that detached from R. norvegicus in the same period (54.8%). A total of nine engorged nymphs (one from R. norvegicus, and eight from C. callosus) were recovered in the infestations. All of them successfully molted to adults, which were all females. The male of I. schulzei remains unknown.

The finding of an unfed adult female of the taiga tick Ixodes persulcatus Schulze is reported from the northern part of Eastern Siberia (the central part of the Sakha Republic [former Yakutia]) of Russia. This finding supplements other reported single findings of the taiga tick in different sites of the central part of the Sakha Republic, thus increasing its distributional range. The reproductive range of the taiga tick is limited to two separate areas in the southern parts of the Republic. The most probable mode of tick introduction northwards from the border of the reproductive range is by spring bird migrations from their wintering areas to breeding sites. The possibility of the establishment of stable tick populations in the areas of introduction is also considered.