We evaluated the effect of holding temperature and time between mosquito death and processing mosquito pools for virus detection on our ability to detect West Nile (WN) viral RNA from pools of mosquitoes by reverse transcriptase-polymerase chain reaction (RT-PCR). Pools of 24 uninfected Culex pipiens L. mosquitoes were “spiked” with either a single Cx. pipiens that had been inoculated previously with WN virus or with an uninfected mosquito. These pools were held dry at 20, 4, −20, or −70°C for selected time intervals before all mosquito pools were triturated in TRIzol®LS reagent and processed for detection of WN viral RNA. While infectious virus virtually disappeared from pools maintained at 20°C by 48 h after mosquito death, neither holding temperature (20 to −70°C) nor holding period (up to 2 wk) affected detection of WN viral RNA by real-time RT-PCR. These findings suggest that we need not keep mosquitoes chilled to be able to detect WN viral RNA effectively by RT-PCR. This should enhance the feasibility of field-based WN virus surveillance programs where only detection of WN viral RNA is the objective and maintenance of a cold chain may not be possible.

Aedes albopictus (Skuse), a mosquito vector of the dengue fever virus, is prevalent in Japan, distributed widely in Honshu Island with its northern limits between latitude 38° to 40°degrees north. The factors affecting distribution of the species in the northern part of Japan were studied using the geographical information system (GIS). During 1998–2000, larval surveillance was carried out in 26 urban and rural areas in the Tohoku district, in the northern part of Honshu Island, by collecting larvae from artificial and natural habitats. Climatological analysis, using the GIS, showed that the following conditions accounted for the current distribution of Ae. albopictus: an annual mean temperature higher than 11°C and a mean temperature of the coldest month, January, higher than −2°C. A period with temperature above 11°C in the confirmed area of the mosquito successively continues for more than 186 d per year. The accumulated temperature calculated from a temperature of 11°C, which may be close to the developmental zero of Ae. albopictus, was over 1,350 degree-days. The relationship between the beginning of short-daylength, inducing egg diapause, and the monthly mean temperature during September and October necessary for successful larval development in the Tohoku district is also discussed. We also show the relationship between the current distribution of Ae. albopictus and the annual mean temperature in the United States. From these results it is predicted that Ae. albpictus will be established in some cities in northeast United States.

Sand fly (Diptera: Phlebotominae) fauna were surveyed in various districts of Sanliurfa in southeast Turkey for 3 yr immediately after an epidemic of cutaneous leishmaniasis (Leishmania tropica). Sticky papers and CDC light traps collected a total of 10,937 sand flies, of which 10,919 (4,158 females and 6,761 males) were identified as Phlebotomus and 18 (11 females and seven males) as Sergentomyia (S. theodori Parrot; S. adleri Theodor). Eight Phlebotomus spp. were identified: P. sergenti Parrot (72.3%), P. papatasi (Scopoli) (27.2%), P. brevis Theodor & Mesghali (0.20%), P. neglectus Leger & Pesson (0.13%), P. perfiliewi Parrot (0.05%), P. mascitti Grassi, P. halepensis Theodor, and P. alexandri Sinton (0.01%). Phlebotomus mascitti and P. neglectus, along with both Sergentomyia sp., have not been previously described from the study area. Similar results were obtained when both trapping methods were applied in the same houses, indicating that local P. sergenti and P. papatasi populations were equally attracted to the light. P. sergenti was consistently abundant, agreeing with the general view that this species is the vector of leishmaniasis in the region. There was no apparent decrease in the relative abundance of this vector versus the other species, suggesting that factor(s) other than a change in the dynamics of sand fly populations precipitated the decline of the human leishmaniasis epidemic in Sanliurfa.

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An. farauti 2, An. koliensis Owen, and An. punctulatus Dönitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.

This article provides taxonomic keys for the identification of the fourth-instar larvae and females of 24 species of anopheline mosquitoes (seven species in subgenus Anopheles and 17 species in subgenus Cellia) recorded from Pakistan. The keys are based on literature sources as well as the examination of field and museum collections.

Sensilla of the antennae of male and female Dermatobia hominis were studied by scanning electron microscopy. The images obtained were interpreted according to the scientific literature referring to the sensory structures of insects. Sensilla of the “long bristle” type and smaller spines of the “microtrichia” type were found in different numbers and patterns of distribution on the scape and pedicel. Coeloconic sensilla with longitudinal cuticular furrows were observed on the female flagellum, as well as two varieties of basiconic sensilla: a large one surrounded by pointed foliaceous structures and a smaller form implanted on a raised cuticular process. The larger type of basiconic sensilla was also observed on the flagellum of the male antenna, as well as a variety of coeloconic sensilla with apical dilatations. Trichoid and chaetic sensilla were encountered in greater numbers on the arista of females, with the former type predominating. Coeloconic sensilla were observed on the arista of both sexes, as well as sensilla of the “long bristle” and styloconic types exclusively in males. Adult D. hominis were observed to possess sensory structures with chemoreceptory, thermo-hygroreceptory and mechanoreceptory functions on their antennae. These results could facilitate the identification of the chemoreceptors by electrophysiological techniques. The sexual dimorphism noted for the antennae constitutes a new criterion for distinguishing between the sexes of D. hominis

Behavioral implications of Trypanosoma cruzi Chagas infection in Rhodnius prolixus Stål were observed. Feeding and defecation behaviors of infected versus uninfected insects were assessed on an artificial membrane-feeding system and on live guinea pigs. Based on a defecation index, fifth instars were the most efficient vectors, followed by adult females, fourth instars, and adult males. Bugs fasted for longer periods (5–6 mo) took smaller blood meals but defecated significantly earlier than bugs fasted for shorter periods (2–3 mo). Multiple blood feeding, degree of fasting, life stage, T. cruzi infection, and gender affected the vector potential of R. prolixus. Our data indicate that T. cruzi and R. prolixus have not coevolved to facilitate the transmission of T. cruzi, which suggests that this parasite-host relationship may be relatively recent.

The internal transcribed spacer (ITS) regions of the ribosomal DNA of house flies, Musca domestica L., the stable flies, Stomoxys calcitrans (L.), and four parasitoid species in the genus Muscidifurax (Hymenoptera: Pteromalidae) were characterized to develop a method based on the polymerase chain reaction (PCR) to better define the role of pteromalid parasitism of pupae of the house fly and stable fly. Two parasitoid-specific primers were designed to anneal to the 5′ end of the 5.8S rRNA gene in the parasitoid species. When paired with a universal primer at the 3′ end of the 18S rRNA, the primers amplified the target ITS1 region in 10 pteromalid species. PCR allowed detection of parasitoid DNA within 24 h after females of Spalangia endius Walker oviposited into house fly puparia. PCR failed to amplify parasitoid DNA or detect parasitism in puparia that were exposed to parasitoid oviposition, allowed to develop 7 d, then killed by freezing and held at 20–24°C for 4 d to allow DNA degradation. Digestion of the PCR products with restriction enzymes produced restriction fragment length polymorphisms that allowed identification of individual parasitoid species. Significantly greater levels of parasitism (P < 0.05) were detected by PCR for two of the five field collection dates in 1997. On the dates when PCR detected higher levels of parasitism than estimates provided by emergence of adult insects from samples taken at Feedlot M in 1997, more than 65% of all puparia in the emergence samples failed to produce an adult insect. Three puparia collected in 1997 produced double PCR bands that corresponded to PCR band sizes of Muscidifurax spp. and Spalangia sp., possibly indicating multiple parasitism or hyperparasitism.

The immune response against different organisms and particles inoculated in the hemocoel of female Anopheles albimanus Wiedemann was investigated. Histological and ultrastructural observations indicated that melanization and hemocyte type participation varied according to the particles inoculated. The initial responses against heat-killed Microccocus lysodeikticus and Escherichia coli included hemocyte lysis and melanization whereas the response to heat-killed Saccharomyces cerevisiae was only cellular, and an initial melanization of Sephadex G-25 (neutral charged) beads was followed by the formation of cellular aggregates. After 24 h, hemocytes were involved in all terminal encapsulation events. Plasmodium vivax Grassi and Feletti formalin-fixed sporozoites induced a weak response. Cellular aggregates were observed 1 h postinoculation, but participating hemocytes could not be identified because of the extensive cellular damage and lysis. Sporozoites were also observed in the core of these aggregates, mixed with cell debris and free in the hemolymph. The effect on the inoculated particles was also different—S. cerevisiae was encapsulated only by hemocytes, whereas M. lysodeikticus was lysed and E. coli was phagocytosed by plasmatocytes. These results indicate that hemocytes are important components in the immune response in An. albimanus.

We studied the larval distribution and composition of Anopheles arabiensis Patton, An. gambiae s.s. Giles, and its forms, among local habitats; and their association with the adults between these habitats in Banambani village, Mali during the mid-rainy seasons of 1997–1999. For species and form identification we used polymerase chain reaction (PCR) and PCR-restriction fragment-length polymorphism (RFLP). Differences among species in the distribution of larvae were observed in 1998, but not in 1997 or 1999, although they were on the borderline of statistical significance. Differences among the M and S molecular forms were statistically significant in 1999 when rainfall was high, but not in the two prior, drier sampling periods. Combining all information into the Fisher multiple comparisons test, there were statistically significant differences between species and molecular forms during the 3-yr study period. Hybrid larvae between the M and S forms were observed (0.57%), the first such observation to our knowledge. In spite of differences among larval distribution, no differences of adult species composition were observed among habitats. Factors that influence the distributions of An. gambiae larval populations are discussed.

In total, 324 Anopheles funestus Giles specimens collected from seven houses in western Kenya and seven in coastal Kenya were scored for their paracentric chromosomal inversions with the aim of determining the level of genetic differentiation based on these inversions. Houses in each area were within a 2-km radius. The two areas are ≈700 km apart. Only inversions 2a, 3a, 3b, and 5a were found to be polymorphic. Levels of polymorphism varied greatly between inversions. There were no significant deviations from Hardy-Weinberg expectations for samples from individual houses at one site or when data for houses in each area were pooled. Overall, the level of differentiation between western and coastal Kenya was significant, suggesting that the two populations are genetically isolated. Results based on inversion 2a alone were, however, not consistent with this conclusion. Founder effects and selection against the 2a inversion are discussed as possible explanations for this discrepancy.

The melanization responses of field-captured Anopheles gambiae s.l. toward oocysts of the malaria parasite Plasmodium falciparum or negatively charged (C-25) Sephadex beads were determined. Only two of 431 infected mosquitoes harboured melanized oocysts. However, 90% of field-captured mosquitoes melanized C-25 Sephadex beads. The effects of age, glucose concentration and blood meal on the melanization response of an An gambiae s.s. laboratory colony toward C-25 beads were also assayed. All newly emerged females (which did not blood-feed) melanized the beads. By 4 d postemergence, there was a marked reduction in melanization response, particularly among those mosquitoes that had not blood fed. A blood meal, however, taken by 4-d-old mosquitoes increased their immune response as did high glucose concentrations in the nonblood-fed group. These data indicate that C-25 Sephadex beads can estimate the general strength of An. gambiae’s immune response. However, C-25 beads do not accurately model An. gambiae’s susceptibility to P. falciparum oocysts in natural populations. To the best of our knowledge, this is the first report of field refractoriness in An. gambiae s.l.

Stable flies, Stomoxys calcitrans (L.), were orally infected with Aeromonas sp., Pseudomonas aeruginosa (Schroeter), and Serratia marcescens Bizio that were cultured on egg-yolk media, nutrient broth, and fly egg media. Aeromonas and Serratia caused mortality when the bacteria were originally grown on egg-yolk medium. Pseudomonas was equally lethal regardless of the media on which it was cultured. A wild isolate of Aeromonas caused greater death than an isolate that had been passed through host flies and had been reisolated from killed flies. Mortality increased with bacterial dose for all species. Aeromonas and Serratia caused mortality within several days after ingestion, whereas Pseudomonas caused a gradual increase in mortality 3–7 d after ingestion. The pathologic activity of Aeromonas and Serratia required extracellular products produced when cells were grown in egg yolk medium. Aeromonas required both supernatant and cells from egg yolk medium, wereas Serratia required supernatant from egg yolk medium and cells from either nutrient broth or egg yolk medium. Mortality due to ingestion of Aeromonas was correlated with the presence of enzymes that cause α- and β-hemolysis, while mortality following ingestion of Serratia was associated with β-hemolysins, elastases, and chitinases.

Two different doses of Ross River virus (RR) were fed to Ochlerotatus vigilax (Skuse), the primary coastal vector in Australia; and blood engorged females were held at different temperatures up to 35 d. After ingesting 10^4.3 CCID₅₀/mosquito, mosquitoes reared at 18 and 25°C (and held at the same temperature) had higher body remnant and head and salivary gland titers than those held at 32°C, although infection rates were comparable. At 18, 25, and 32°C, respectively, virus was first detected in the salivary glands on days 3, 2, and 3. Based on a previously demonstrated 98.7% concordance between salivary gland infection and transmission, the extrinsic incubation periods were estimated as 5, 4, and 3 d, respectively, for these three temperatures. When Oc. vigilax reared at 18, 25, or 32°C were fed a lower dosage of 10^3.3 CCID₅₀ RR/mosquito, and assayed after 7 d extrinsic incubation at these (or combinations of these) temperatures, infection rates and titers were similar. However, by 14 d, infection rates and titers of those reared and held at 18 and 32°C were significantly higher and lower, respectively. However, this process was reversible when the moderate 25°C was involved, and intermediate infection rates and titers resulted. These data indicate that for the strains of RR and Oc. vigilax used, rearing temperature is unimportant to vector competence in the field, and that ambient temperature variations will modulate or enhance detectable infection rates only after 7 d extrinsic incubation. Because of the short duration of extrinsic incubation, however, this will do little to influence RR epidemiology, because by this time some Oc. vigilax could be seeking their third blood meal, the latter two being infectious.

Seasonal dispersal of Carcinops pumilio (Erichson) was evaluated using two trapping methods—a black-light pitfall trap and a mesh-bottomed trap placed on poultry manure. The black-light trap collected larger numbers than the mesh-bottomed trap from March through June. The mesh-bottomed trap gathered larger numbers of beetles from June through August and numbers were less variable throughout the year. Often, when very low numbers of beetles were recovered from manure cores, large numbers of beetles could be collected with the black-light trap suggesting that beetle density may not be an important factor in dispersal behavior. The greatest dispersal in the dispersal arenas (≈90%) occurred using beetles collected by both trap types in June 2000. Beginning in March and ending in August, a cyclic rise and then fall pattern in both laboratory dispersal and beetle collections was observed. Trap collection patterns were similar in both years of the study. In January and March, we were unable to prevent dispersal behavior of beetles captured in black-light traps. However, in May, after beetles had been in a dispersal phase for several months, we were able to suppress dispersal. In contrast, dispersal behavior among beetles captured with the mesh-bottomed trap did not change following the photoperiod-altered exposure.

From January 1998 through September 1999, 324 dogs in three northwestern Georgia counties were examined for ticks. Six species of ticks were recovered. The three most commonly collected ticks were the American dog tick, Dermacentor variabilis (Say) (310 ♂♂, 352♀♀; prevalence, 97%; mean intensity 2.1); the brown dog tick, Rhipicephalus sanguineus (Latreille) (118♂♂, 119♀♀, 38 nymphs; prevalence, 22%; mean intensity, 3.8); and the lone star tick, Amblyomma americanum (L.) (8♂♂, 26♀♀, 2 nymphs; prevalence, 5%; mean intensity, 2.4). Other ticks recovered were Ixodes cookei Packard (3♀♀); the Gulf Coast tick, Amblyomma maculatum Koch (2♂♂); and the blacklegged tick, Ixodes scapularis Say (1♀). Another adult female specimen of I. scapularis was recovered from a cat, further reinforcing that this medically important tick is present in northwestern Georgia.

We measured the abundance of mosquitoes [primarily Aedes vexans (Meigen) and Culex tarsalis Coquillett] at cliff swallow (Petrochelidon pyrrhonota Vieillot) colonies of different sizes in southwestern Nebraska in 1999. Using CO₂ traps placed inside and outside of colonies, we found that total mosquito abundance increased significantly with the number of active cliff swallow nests at a colony site. We found no effect of date or weather conditions on the number of mosquitoes caught at the different sites. By classifying the landscape from aerial photographs within a 2-km-diameter circle centered on each colony site, we found no significant relationships between habitat type near a colony site and cliff swallow colony size or mosquito abundance. Proximity to livestock could not account for our results. Culex tarsalis was proportionately more likely to be caught inside a colony than at traps 30 m away, but the proportion of C. tarsalis inside a colony did not vary with colony size. Our results cannot be explained by date- or weather-related sampling artifacts or by differences in habitat between sites. Most likely, mosquitoes, especially A. vexans, are attracted to the vicinity of large cliff swallow colonies.

In laboratory tests gravid female Toxorhynchites moctezuma (Dyar & Knab) and Toxorhynchites amboinensis (Doleschall) were offered the choice of black oviposition jars containing a diethyl ether extract of water collected from a natural oviposition site for these species (i.e., used tires), a dilution series of 4-methylcyclohexanol, 3-methylindole, 2-methylphenol, 3-methylphenol and 4-methylphenol, or solvent. Tire water extract and all test compounds acted as oviposition attractants and stimulants for both species, with the threshold amounts required to elicit these behaviors varying between the species and among the compounds tested.

The attractiveness of inflated beach balls covered with adhesive and used as decoys to trap adult stable flies was investigated on Florida panhandle beaches. Decoys were painted either solid black, solid white, or a mixed pattern that consisted of three equally spaced white circles (20 cm diameter) on a solid black background. Another set of decoys (referred to as plain) were unpainted and retained the manufacturer’s original color scheme. The plain decoy consisted of a separate blue, yellow, and red diamond-shaped panel. Each color panel was separated by a white panel of similar size and orientation. Plain decoys collected significantly (<0.05) more stable flies than other treatments but no significant difference was noted between colored panels. The mixed pattern decoy captured significantly fewer flies than the plain decoy but significantly more flies (nearly twice) when compared with solid white or black decoys. No difference in preference was observed when fly abundance on the black background was compared with that on white circles and total abundance from both areas appeared to be additive compared with either area alone. No significant differences were found in the number of flies trapped on solid white versus black decoys. When trapping efficiency was compared with Alsynite translucent fiberglass cylinders covered with adhesive-treated cellophane sheets, the decoy trap caught significantly more (>10 times) flies per square centimeter. Alsynite cylinders are considered standard tools when sampling fly populations. Adhesive-treated beach ball decoys may be an alternative method for luring stable flies away from human hosts in recreation areas, or from animals, thereby reducing biting annoyance from these pests.

A simple bioassay system was developed to study locomotory and ovipositional responses of screwworm, Cochliomyia hominivorax (Coquerel), flies to bovine blood inoculated with eight species of coliform bacteria that were isolated from screwworm-infested animal wounds. When exposed to odors from bacteria-inoculated blood which was incubated for 72 h at 37°C, ≈50% of 7- and 10-d-old gravid females landed on the blood by the end of 15 min test exposure. Only 17% of 7-d-old reproductively sterile females (from irradiated pupae) with previtellogenic ovaries and 2% of 4-d-old vitellogenic females responded to the same treatment. Females generally reacted in greatest numbers to bacteria-inoculated blood incubated for 72 h, followed by 48 h, then 24 and 96 h. Males of all ages tested were unresponsive. Although oviposition occurred in tests with gravid females lasting for 1 h, with both inoculated blood and an uninoculated control, the inoculated sample was significantly better than the control at 48, 72, and 96 h incubation duration. Our results are consistent with the conclusion that the inoculated blood, when incubated for 48–72 h, gives off volatile chemicals which attract gravid females and contains an oviposition stimulant that acts following contact and feeding. The volatiles, once isolated and identified, may be useful for sampling gravid females in the field as well as improving the oviposition system in the mass-production facility of the screwworm eradication program.

Ixodes didelphidis Fonseca & Aragão was described in Brazil in 1952 as a new tick species that differed from Ixodes loricatus Neumann by the spiracular plate pattern. We have reared four tick colonies from different geographic areas in the laboratory that were started from single engorged females originally identified as I. didelphidis (BMG colony) and I. loricatus (CSP, PSP, and TRJ colonies). We analyzed the spiracular plate morphology of F₁ adult ticks from each tick colony, compared their biological data, and performed a molecular analysis of the second internal transcribed rDNA spacer (ITS2) to test the validity of the species I. didelphidis. The spiracular plate analysis of laboratory F₁ adult ticks yielded from single females from the four colonies showed variations that invalidate morphological parameters for differentiation of I. loricatus and I. didelphidis. Biological data of the BMG, CSP, and TRJ colonies were similar. The biology of the PSP colony was not evaluated. The ITS2 sequence variations observed between the tick colonies ranged from 1.3 to 4.9%, and the similarity tree constructed by the neighbor-joining method with nucleotide distances showed that the distances between the samples were similar to what is expected for intraspecific variations found in other ticks species. The morphological and biological results, in conjunction with the ITS2 analysis, supported the conspecificity of I. loricatus and I. didelphidis.

RESUMO Ixodes didelphidis Fonseca e Aragão foi descrito no Brazil em 1952 como uma nova espécie de carrapato que se diferenciava de Ixodes loricatus Neumann através do padrão da placa espiracular. Nós criamos em laboratório quatro colônias de carrapatos de diferentes áreas geográficas, iniciadas a partir de fêmeas ingurgitadas originalmente identificadas como I. didelphidis (colônia BMG) e I. loricatus (colônias CSP, PSP, e TRJ). Nós analisamos a morfologia da placa espiracular dos adultos F₁ de cada colônia, comparamos seus dados biológicos e fizemos uma análise molecular do segundo espaço transcrito interno do rDNA (ITS2) para verificar a validade da espécie I. didelphidis. A análise da placa espiracular de adultos provenientes de uma mesma fêmea de cada uma das colônias demonstrou resultados controversos que invalidam os parâmetros morfológicos publicados para diferenciação de I. loricatus e I. didelphidis. Os dados biológicos das colônias BMG, CSP, e TRJ foram similares. As variações nas seqüências do ITS2 entre as colônias de carrapatos (amplitude: 1,3–4,9%) e a árvore de similaridade com as distâncias de nucleotídeos mostraram que as distâncias entre as amostras foram similares ao esperado para variações intra-específicas encontradas em outras espécies de carrapatos. Os resultados morfológicos, biológicos e moleculares fornecem evidências para considerarmos I. loricatus e I. didelphidis como a mesma espécie.

The objective of this study was to determine the epidemiological significance of subterranean mosquito breeding sites to the 1993 outbreak of dengue fever (type 2) in the northern Queensland town of Charters Towers, Australia. In recent studies on subterranean mosquito breeding, containers such as wells and service manholes have been shown to be important breeding sites to Australia’s only dengue vector, Aedes aegypti (L.). This study demonstrates a direct epidemiological association between subterranean breeding sites and dengue virus infection. The mean distance between residents seropositive for dengue 2 and the nearest subterranean container (113 m) was significantly less than for a randomly selected control (191 m), (F = 81.9; df = 1, 478; P < 0.001). Residents positive for dengue 2 antibodies was 2.47 (95% confidence interval 1.88–3.24) times higher for those living within 160 m of a well or service manhole, compared with those residing further away. These findings emphasize the importance of including subterranean water containers in Ae. aegypti surveillance and control programs.

The susceptibility of three anopheline mosquitoes, Anopheles minimus Theobald, An. sinensis Wiedemann, and An. saperoi Bohart & Ingram, from the Ryukyu Archipelago to the rodent malaria, Plasmodium yoelii nigeriense was examined to find new vectors other than An. stephensi Liston for rodent malaria studies in the laboratory. The survival rate of the mosquitoes after feeding on mice infected with P. y. nigeriense was also examined. The Beech strain of An. stephensi from India was compared with An. minimus from Ishigaki Island, and An. sinensis and An. saperoi from Okinawa Island. Oocysts were first found on day 3 after feeding on mice infected with P. y. nigeriense in An. stephensi, on day 4 in An. minimus and An. saperoi, and day 6 in An. sinensis. From 8 to 14 d after feeding on malaria-positive mice, oocysts were present in 97.2–100% of An. stephensi, 85.7–100% of An. saperoi, 20–74.1% of An. minimus, and 12.5–13.3% of An. sinensis. The duration of oocyst occurrence in An. saperoi was 55 d, the longest among the anopheline mosquitoes used in this study. On day 8 after feeding, sporozoites were found in the salivary glands and heads of all the mosquitoes tested. From the 10th to 16th d, sporozoites were present in the salivary glands of 14.9% (range, 9.1–28.0%) of An. minimus, 47.3% (40.7–58.1) of An. saperoi, and 96.2% (94.1–97.2) of An. stephensi, but were absent in An. sinensis. Anopheles saperoi could be an excellent vector of P. y. nigeriense because it has comparatively high susceptibility and high longevity even after feeding on infected mice.

Two insecticides, fipronil and imidacloprid, were evaluated for efficacy and longevity against Oropsylla montana (Baker), the most important vector of plague in California. Wild-caught California ground squirrels, Spermophilus beecheyi (Richardson), were individually housed in the laboratory to serve as natural hosts to O. montana and for on-animal insecticide trials. Several concentrations of technical grade fipronil and imidacloprid in acetone were applied to samples of clean rodent bedding to determine residual activity and longevity against fleas. Immature and adult cat fleas, Ctenocephalides felis (Bouché), were used as representative fleas for periodic assays in place of less fecund O. montana. Toxicity of treated bedding did not decrease significantly for 1 yr at all applied concentrations. Fipronil provided 100% kill for at least 1 yr at ≥100 ppm, whereas imidacloprid required 10,000 ppm for similar performance. Laboratory squirrels were treated with topical formulations of fipronil (Frontline Top Spot) and imidacloprid (Advantage Flea Adulticide) at a dosage rate of 15 mg/kg and evaluated for residual activity every 2 wk against adult O. montana. Residual activity was determined by percent recovery of O. montana adults released on treated and untreated animals after 48 h. Frontline provided 100% kill of adult fleas for at least 10 wk, and up to 26 wk on one animal. Advantage failed to provide 100% kill of adult fleas at 2 wk, with complete loss of efficacy by week 6. Concurrent assays with bedding samples from squirrel nest boxes showed negligible toxicity transfer from treated animals to nest bedding.

The growth and development of Anopheles gambiae Giles larvae were studied in artificial habitats in western Kenya. Larvae responded to increasing densities by extending their development time and by emerging as smaller adults, although survival was not significantly affected. Addition of nutrients in the form of cow dung collected near the study site had no impact on larval growth and development. Regression analysis showed that female development time increased by 0.020 d and female dry mass decreased by 0.74 μg with each additional larva. By fitting the data to the pupation window model, the estimated minimum dry mass to achieve pupation was 0.130 mg and the estimated minimum time to pupation was 5 d. The most likely food source for An. gambiae larvae was algal growth, which was significantly reduced by the presence of larvae. Bacterial densities were not significantly affected by the presence of larvae although total bacteria counts were lower at the higher densities indicating they may provide a secondary food source when algal resources are depleted. Similarly, the levels of nitrogen and phosphorus in the habitats were not significantly affected by the presence of larvae although there was evidence of decreasing nitrogen levels occurring with increasing larval densities suggesting that nitrogen may be a limiting resource in the larval environment. The data indicate that competition within the larval environment may indirectly regulate An. gambiae populations by reducing adult body size, which may in turn reduce adult survivorship and fecundity. The potential impact of density-dependent interactions among An. gambiae larvae on the transmission of Plasmodium falciparum is discussed.

To investigate the immunological mechanisms of acquired resistance to tick infestation, interferon gamma (IFN-γ) deficient mice (IFN-γ −/− mice) were used to assess interleukin-4 (IL-4) and antibody production levels against tick salivary gland antigen on three successive infestations with Haemaphysalis longicornis Neumann nymphs. The engorged body weight of the ticks decreased during the second and third infestations. Similar observations were noted in IFN-γ / mice. However, the engorged body weight of the ticks from IFN-γ / mice were considerably lower than those from IFN-γ −/− mice. A marked increase in antibody production during the second and third infestations was observed indicating that IFN-γ −/− mice could acquire immunological resistance against H. longicornis nymphs. Moreover, IL-4 levels were higher during the first and third infestations but decreased during the second infestation. IL-4 levels were significantly higher in IFN-γ −/− mice than in IFN-γ / mice. We have shown here that the statistically significant high IL-4 levels observed in IFN-γ −/− mice may be a result of type 2 helper cell (Th2) polarization. However, the apparently higher IL-4 levels during the first and third infestations and the notable decline during the second infestation suggest that other cytokines or factors in the host immune system may play a part in regulating IL-4 levels.

Borrelia burgdorferi was found widespread in ixodid ticks from the Basque Country (Spain) during a two-step study. In the first part, a total of 7,835 ixodids of eight different species was collected from vegetation, classified, and processed using polymerase chain reaction (PCR) for detection of B. burgdorferi ospA DNA. B. burgdorferi DNA was detected in ≤12.5% of adults and ≥0.6% of Ixodes ricinus (L., 1758) nymphs (mean 1.5 and 0.05%, respectively), and in ≤14.3% of adult Hemaphysalis punctata (Canestrini & Fanzago, 1877) analyzed (mean 1.2%). The second part of the study was undertaken 2 yr later to characterize B. burgdorferi distribution by focusing on the areas where I. ricinus was the predominant species. Ten areas were selected from which 1,535 nymphs and adults of I. ricinus were collected and processed by PCR and culture techniques. Infected ticks were found in all zones. B. burgdorferi DNA was detected in a mean of 9.3 and 1.5% of adults and nymphs, respectively. Nine isolates of B. burgdorferi were obtained, belonging to four different genospecies (B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. lusitaniae). The results indicate that some areas of Spain have a potential risk for Lyme disease agent exposure and that B. burgdorferi appears to have an increasing occurrence in ticks in the Basque Country.

The egg hatchability, insemination, and longevity of Japanese Culex pipiens pallens Coquillett and Japanese Culex quinquefasciatus Say were compared at 25 and 30°C. Egg hatchability was high in Cx. p. pallens at 25°C, but it was very low at 30°C because almost no females were inseminated at this temperature. In Cx. quinquefasciatus, the egg hatchability and insemination rates were very high, even at 30°C. The longevity of adult females and males was generally shorter in Cx. p. pallens than in Cx. quinquefasciatus at both temperatures. Because high temperatures may restrict the spread of Cx. p. pallens, we suggest that even if this species spreads to Okinawa, the possibility of it becoming established is very low.

Volatiles emitted by male and female T. infestans before and during copula were collected on Porapak-Q filters, desorbed with dichloromethane, and analyzed by gas chromotography and gas chromatography-mass spectrometry after confirmation of attractiveness in an arena bioassay. Chemical analysis confirmed the presence of (R,S)-2- and 3-methylbutan-1-ol in a 2:1 ratio; short chain acids (ethanoic to nonanoic acid); long chain acids decanoic to (Z)-9-octadecenoic acid; aliphatic aldehydes (hexanal to nonanal), benzaldehyde and dipropylsulphide from insects in copula. Electroantennographic studies conducted with a homologous series of aliphatic aldehydes on female and male T. infestans showed that, for a given dose, EAG responses elicited from both sexes increased with increased chain length up to nonanal, after which EAG-activity declined. Attractiveness of non-acidic trace components identified in the volatiles were tested on male and female T. infestans, in an arena bioassay using a video tracking method. Aliphatic C₆ to C₁₀ aldehydes were tested: hexanal (1–100 μg) and heptanal (10 μg) were attractive to female T. infestans, high doses of octanal and nonanal (1–100 μg) were unattractive to male and female T. infestans but low doses of nonanal (0.01–0.1 μg) were attractive to male T. infestans. Benzaldehyde was highly attractive to female T. infestans at low doses (0.05–0.1 μg). 3-methylbutan-1-ol was attractive to male T. infestans at high dose (1,000 μg). (S) or (S,R) 2-methyl-butan-1-ol were anattractive to males or females (1–1,000 μg). Blends of hexanal and benzaldehyde (20:1 and 40:1) showed an additive effect on attraction compared with hexanal alone, when tested on female T. infestans. The study has demonstrated the presence of a number of electrophysiologically and behaviorally active compounds in volatiles emitted by T. infestans in copula that may have a role in the postulated copulation pheromone.

Tissues of rodents and host-seeking adult ticks collected in the Piedmont, Sandhills, Coastal Plain, and Coastal Zone of South Carolina were cultured in attempts to isolate Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt & Brenner), the etiologic agent of Lyme disease. An exploratory, tree-based statistical analysis was used to identify ecological variables that were associated with spirochete infection among rodents and ticks. Spirochetes were isolated from tissues of 71 rodents: 22 (69%) of 32 eastern woodrats, 39 (53%) of 74 cotton mice, and 11 (25%) of 44 hispid cotton rats. Rodent infection prevalences were significantly higher in the Coastal Zone than in other regions. Spirochetes were also cultured from 31 (2.6%) of 1,193 questing ticks. Prevalence of spirochetes in Ixodes affinis Neumann (19/74, 26%) was significantly higher than in I. scapularis Say (12/864, 1.3%) and other species (0/255) of ticks tested. In addition, two (9%) of 23 adult I. minor Neumann removed from woodrats contained spirochetes. Isolates from rodents and ticks were analyzed immunologically by indirect immunofluorescence and Western blots, and further characterized by polymerase chain reaction assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All were determined to be B. burgdorferi sensu lato. Results of this study confirmed that B. burgdorferi is endemic in South Carolina, and that enzootic transmission cycles exist at foci in the Coastal Zone. These findings add additional evidence that I. affinis and I. minor are potentially significant maintenance vectors of the spirochete.

Two subcellular fractions from the midgut of the malaria mosquito Anopheles stephensi (Liston) were used to immunize BALB/c mice. Mice were subsequently infected with the rodent malaria parasite Plasmodium berghei (Vincke & Lips), and the effects of anti-mosquito immunity on mosquito survival and fecundity and on parasite transmission were investigated. Mosquitoes were infected directly from mice (in vivo) or by feeding cultured ookinetes through a membrane (in vitro). Infections were monitored by counting oocysts on the midgut wall. Microvilli extracts induced a strong and partially specific antibody reaction against the midgut, which was manifest as decreased survival in in vivo fed mosquitoes and reduced fecundity in both kinds of feeding. Antisera against microvilli reduced the mean intensity of P. berghei oocysts when fed in vitro, while mosquitoes fed antiserum against basolateral plasma membranes in vivo, showed higher oocyst burdens.

Mosquitoes, Mansonia indiana Edwards, 1930, were collected from non-endemic area of human lymphatic filariasis and tested for their susceptibility of infection using nocturnally subperiodic Brugia malayiBuckley & Edeson, 1956. Three cats naturally infected with B. malayi were used in the experiment for mosquitoes feeding. The data revealed that the susceptibility of mosquito infection ranged from 30 to 70%. The results also revealed that the susceptibility rates were not linearly correlated to the microfilarial densities in the cat at the time of feeding. The microfilarial density in cats ranged from 15 to 27 per 10 μl of blood whereas the mean number of third stage larvae in the infective mosquitoes ranged from 21.6 to 26.8. In addition, statistical analysis showed no significant difference (P > 0.05) between the mean number of third-stage larvae in mosquitoes and the density of microfilaria in cats. The study indicated that Ma. indiana, collected from non-endemic areas, is capable for transmitting the nocturnally subperiodic B. malayi.

We tested 103 adult Ixodes scapularis Say from 12 counties in Minnesota for the presence of Borrelia burgdorferi and the causative agent of human granulocytic ehrlichiosis (HGE), using polymerase chain reaction (PCR). A total of 17 ticks (16.5%) was positive for B. burgdorferi using nested PCR for the flagellin gene, or both PCR for the ospA gene and nested PCR for the flagellin gene. A total of four ticks (3.8%) was positive for the agent of HGE using nested PCR for 16S rDNA. Counties in Minnesota with established and recently reported populations of I. scapularis both had ticks infected with B. burgdorferi. The agent of HGE was only detected in counties with established I. scapularis populations.

Environmental temperature can affect the ability of mosquitoes to transmit an arbovirus. However, results of various studies indicate that these effects are not consistent among viruses or mosquito species, and there is no information available on the effect of environmental temperature on the ability of North American mosquito species to transmit West Nile (WN) virus. We evaluated the effect of incubation temperature (18, 20, 26, or 30°C) on the ability of Culex pipiens L. derived from specimens collected during the outbreak in New York in 1999 to transmit a strain of WN virus obtained from a crow that died during this outbreak. Although mosquitoes fed on the same viremic chickens, infection rates were directly related to subsequent incubation temperatures. In mosquitoes held at 30°C, virus was recovered from nearly all mosquitoes tested, disseminated infections were detected as early as 4 d after the infectious blood meal, and >90% of all mosquitoes had a disseminated infection 12 or more days after the infectious blood meal. In contrast, for mosquitoes held at 18°C, disseminated infections were not detected until 25 d after the infectious blood meal, and even after 28 d, <30% contained a disseminated infection. Results for mosquitoes held at 20 and 26°C were intermediate for both infection and dissemination rates. The effect of environmental temperature should to be considered when evaluating the vector competence of these mosquitoes and modeling risk of WN virus transmission in nature.

Female Culex tarsalis Coquillett in reproductive diapause were infected per os or by intrathoracic inoculation with western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses during “fall,” maintained over a simulated “winter,” and then tested for virus infection and transmission in vitro and in vivo after “vernal” termination. Exposure of F₁ progeny of field-collected females to cool temperatures and short daylength produced females in reproductive diapause that were reluctant to imbibe infectious virus from pledgets soaked with suspensions of virus, blood and sucrose (2.5% by volume). Those infected per os maintained virus at very low or undetectable titers. Some females that originally tested negative for WEE by plaque assay on Vero cell culture tested positive by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Vero cell culture after passage in mosquito cells. Few females became infected orally with SLE, but these infected females developed elevated titers. Females inoculated with SLE retained their infection through winter and then transmitted readily in vitro and in vivo. Feeding on a vertebrate host after diapause termination significantly increased the titer of SLE in previously infected females. These experiments simulated how infections acquired either horizontally or vertically may provide mechanisms for WEE and SLE overwintering. Attempts to detect infected females during winter following a summer with enzootic WEE activity were negative by both RT-PCR and plaque assay.

Nymphs of Ixodes ricinus (Linnaeus, 1758), I. scapularis Say, 1821, and I. pacificus Cooley & Kohls, 1943 are epidemiologically the most dangerous stage for transmission of Lyme disease to humans. Many factors play a role in the epidemiological significance of the nymphs. In this study, we address the question of whether nymphs show a greater tendency than adults to accept humans as their host. To evaluate this, we have compared the host acceptance behavior of nymphs and adults (males and females) with respect to a human in Rambouillet forest, a focus of Lyme disease. Individual ticks (nymph, male or female) located on a herbaceous stem were exposed to different stimuli (e.g., approach, stem movement, breathing), and the response of each individual to these stimuli was noted. Tick responses were categorised into classes (from 0 to 3) according to their intensity. Statistical analysis carried out on 22 ticks allowed us to compare the behavior of the nymph stage with respect to males and females. Despite the small sample sizes, it appears that nymphs are more responsive to a human than are the adults.

Trunks of 83 trees in a mixed deciduous forests in Maryland were sampled for the presence of nymphs of the blacklegged tick, Ixodes scapularis Say, and the lone star tick, Amblyomma americanum (L.). Although one or more nymphs of either I. scapularis or A. americanum was found in leaf litter and substrate ≤1 m from the bases of 47% of the trees sampled, a total of 6 I. scapularis nymphs was found on the trunks of only five trees. No nymphs were found on the trunks of 12 dead trees. No A. americanum nymphs were found on any tree trunks. The trunks were sampled to 2.5 m above the soil, but the nymphs were found ≤1 m from the ground. More than 50% of I. scapularis nymphs found in the leaf litter ≤1 m from bases of living trees were north of the trees sampled, whereas few I. scapularis were found west of trees. These findings suggest that the I. scapularis nymphs’ presence on tree trunks is of little ecological consequence, unless nymphs were being removed from tree trunks by acquiring hosts at such a rapid rate that nymphal numbers on trunks could not accrue.

In January 2001, while conducting a survey of the tick fauna of the State of Rondônia, Brazil, a rural area within Monte Negro county was visited. On one farm within the county the producer maintained a herd of crossbred swine, Sus scrofa L., that was reared under unconfined conditions, with unrestrained access to the pasture and adjacent native Amazon equatorial forest. Inspection of the swine herd produced a total of 77 ticks collected from eight adult pigs (mean, 9.6 ticks per pig) that were identified as Amblyomma naponense (Packard) (26 males, eight females), A. oblongoguttatum Koch (five males, three females), A. ovale Koch (one female) and A. scalpturatum Neumann (one male). One Amblyomma larva and 32 Amblyomma nymphs also were collected from the pigs. Of these, six nymphs were reared in the laboratory until they reached the adult stage, one being an A. oblongoguttatum female and five being A. scalpturatum females.

As part of an on-going malaria surveillance effort conducted by the U.S. Forces Korea, Republic of Korea (ROK), a total of 28,286 anopheline mosquitoes was tested for the presence of Plasmodium vivax circumsporozoite (CS) protein. Mosquitoes were collected (using a variety of light and baited traps) from 29 locations throughout the ROK (the majority were collected near the de-militarized zone), identified to species, and tested by enzyme-linked immunosorbent assay for the presence of P. vivax 210 and P. vivax 247 CS protein. Recent evidence suggests that characters used to separate Anopheles sinensis Wiedemann from An. lesteri Baisas & Hu are unreliable; therefore, the data have been analyzed by grouping these two species. A total of 25,365 Anopheles sinensis/lesteri, 2,890 An. yatsushiroensis Miyazaki, and 31 An. sineroides Yamada was tested. Of these, one pool of 10 An. sinensis/lesteri collected on 9 September 1999 at Camp Howze and one pool of nine An. sinensis/lesteri collected on 13 September 1999 at Camp Bonifas were positive for P. vivax 247.

As part of an evaluation of potential vectors of arboviruses during a Rift Valley fever (RVF) outbreak in the Nile Valley of Egypt in August 1993, we collected mosquitoes in villages with known RVF viral activity. Mosquitoes were sorted to species, pooled, and processed for virus isolation both by intracerebral inoculation into suckling mice and by inoculation into cell culture. A total of 33 virus isolates was made from 36,024 mosquitoes. Viruses were initially identified by indirect fluorescent antibody testing and consisted of 30 flaviviruses (all members of the Japanese encephalitis complex, most probably West Nile [WN] virus) and three alphaviruses (all members of western equine encephalitis complex, most probably Sindbis). The identity of selected viruses was confirmed by reverse transcriptase-polymerase chain reaction and sequencing. Culex antennatus (Becker) and Culex perexiguus Theobald accounted for five (17%) and 23 (77%) of the WN virus isolations, respectively. Despite isolation of viruses from 32 pools of mosquitoes (both WN and Sindbis viruses were isolated from a single pool), RVF virus was not isolated from these mosquitoes, even though most of them are known competent vectors collected during an ongoing RVF outbreak. Thus, it should be remembered, that even during a known arbovirus outbreak, other arboviruses may still be circulating and causing disease.