A blinded laboratory evaluation compared the accuracy, sensitivity, and specificity of an in situ enzyme immunoassay (EIA), VecTest wicking assay, and reverse transcription-polymerase chain reaction (RT-PCR) to detect and distinguish West Nile (WN) and St. Louis encephalitis (SLE) viruses in pools of 50 mosquitoes. Adult female Culex tarsalis Coquillett were inoculated with either WN or SLE viruses, held for 0–11 d at 28°C, killed by freezing, and then were added to 49 or 48 uninfected mosquitoes to make up 14 pools positive for WN virus, 14 positive for SLE virus, 14 positive for both WN and SLE viruses, and 14 negative for virus. Pools were number coded and tested blindly. Virus was not detected in known negative pools. VecTest and RT-PCR assays were comparably sensitive and accurate, detecting virus in pools containing females held for 3 d postinoculation; only RT-PCR detected SLE virus in pools on days 0–1. The VecTest and RT-PCR produced a single false-positive result for WN and SLE, respectively. RT-PCR detected RNA in samples positive by the VecTest, indicating that the detergent in the wicking buffer did not prevent RT-PCR from confirming VecTest results. Detector antibodies used in the in situ EIA cross-reacted between SLE and WN viruses, reducing accuracy. Both the VecTest and RT-PCR provided rapid and specific results, but they detected only those viruses known to be present. Plaque assay on Vero cells was comparably sensitive and had the added benefit of detecting newly emerging viruses, but this method required virus culture followed by identification, thereby delaying reporting.

Antennal sensilla of the Cochliomyia hominivorax were studied by scanning electron microscopy. Thirteen subtypes of sensilla were identified in the fly antenna. In the antennal funiculus, nine subtypes were found: one trichoid, three basiconic, and five coeloconic. Among these, olfactory sensilla were morphologically recognized. Long bristle sensilla presenting various sizes and distribution patterns were observed in the scape and pedicel. In this segment, two other types of tricoid sensilla were still observed. The three antennal segments were shown to be coved by smaller spinules of the microtrichia type. No type of sensillum was seen in the aristas of C. hominivorax. Consistent differences between the sexes in typology, topography, and quantity of antennal sensilla were verified. The great variety of sensillary subtypes, presenting wall pores and/or grooves, found in the funiculus of C. hominivorax, indicates the olfaction should play an important role in this species behavior and communication. The data of the current study, besides corroborating in the amplification of the morphologic and ultrastructural knowledge of C. hominivorax, can foment future electrophysiological studies for the development of volatile semiochemicals, to be uses as attractants in traps, as a new alternative to monitoring and/or controlling this fly.

The surfaces of the body segments and appendages of male and female Dermatobia hominis (L. Jr., 1781) were studied by scanning electron microscopy with the objective of contributing to the current knowledge of the morphology of this insect. The image analyses were done with consideration of the scientific literature on morphology, ultrastructure, and insect sensory structures. A new criterion for distinguishing the sexes of this fly was observed as well as a new setiform empodium type associated with other smaller setae. Different types of trichoid sensilla were observed, as well as spinules, a type of cuticular setiform projection and a scale-like structure. The possible function of the sensilla was discussed, and sensilla with mechanosensory and chemosensory functions were found. Pilose regions that could be involved in the production and/or liberation of pheromones were encountered in the intersegmental spaces of the female abdomen. The current study of the ultrastructure of the body surface of D. hominis contributes to current knowledge of morphology, taxonomy, and sensory structures of this species.

Mosquito species of the Anopheles nili group (Diptera: Culicidae) transmit malaria to humans along rivers in Africa. To date, the An. nili group includes the species Anopheles nili s.s. and its pale-winged variant known as the “Congo form,” Anopheles somalicus and Anopheles carnevalei. Larval and adult mosquito collections in the forest region of Campo, in southern Cameroon, uncovered an additional morphological variant provisionally called “Oveng form” that was subsequently found to be genetically distinct from the other members of the An. nili group. In this study, we provide further biological data that characterizes this new taxon and justifies elevation to specific rank. We propose calling this new species Anopheles ovengensis, after its geographical origin. We present a morphological description of the adult female and fourth instars and original data on the biology, ecology, and role as a human malaria vector of this new species in its type location. We provide dichotomous keys for identification of adult females and fourth instars that can be used at least in tropical areas of west and central Africa.

The salivary glands of male and female Toxorhynchites splendens have the same morphology, and they are paired organs lying on either side of the esophagus. Each gland is composed of two identical tubular lobes, joined together at the end of the proximal region. In the gland, a salivary duct extends through the length of each lobe. The general cellular architecture of the salivary gland of this mosquito is unique. No secretory cavity was found in any cell, and the salivary materials are secreted from long microvilli and collect in a periductal space surrounding the duct. In addition, a number of mitochondria, rough endoplasmic reticulum, and a very large nucleus were observed, suggesting a high energy requirement for producing the salivary proteins involved in sugar feeding. The size of the gland is ≈50 μm in diameter and 1.5 mm in length. These dimensions correlate with high protein content of these salivary glands (2.88 ± 0.14 μg/gland pair). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic protein profiles of the male and female salivary glands were identical. No dominant major proteins were found. Compared with Aedes and Anopheles mosquitoes, the protein profile of T. splendens was similar to that observed in the males of these other species but different to that shown by the females, thus making T. splendens an excellent organism for studying the biochemistry of sugar feeding in mosquitoes.

Members of the New World mosquito subgenus Melanoconion of the genus Culex are important vectors of many alphaviruses including eastern and Venezuelan equine encephalitis viruses (VEEV). We investigated the phylogenetic relationships among nine putative species of the Vomerifer and Pedroi Groups of the Neotropical Spissipes Section by sequencing the internal transcribed spacer 2 (ITS-2) region of ribosomal DNA and using phylogenetic analyses. Results demonstrated that, within the Spissipes Section, the Vomerifer and Pedroi Groups are monophyletic sister groups. The clade comprised by Culex adamesi and Culex ribeirensis showed a sister group relationship to the group consisting of Culex pedroi sequences. The monophyly of the Vomerifer Group corroborated previous suggestions that it is a natural group. However, our topology showed that there are two well-supported, divergent groups within a major clade consisting of Cx. pedroi sequences, suggesting the possibility of a cryptic Cx. pedroi–like species. This finding could have important epidemiological implications for VEEV transmission in Central and South America.

We evaluated the internal transcribed spacer two (ITS2) sequence to detect intraspecific polymorphism in the Palearctic Anopheles maculipennis complex, analyzing 52 populations from 12 countries and representing six species. For An. messeae, two fragments of the cytochrome oxidase I (COI) gene were also evaluated. The results were compared with GenBank sequences and data from the literature. ITS2 analysis revealed evident intraspecific polymorphism for An. messeae and a slightly less evident polymorphism for An. melanoon, whereas for each of the other species, 100% identity was found among populations. ITS2 analysis of An. messeae identified five haplotypes that were consistent with the geographical origin of the populations. ITS2 seems to be a reliable marker of intraspecific polymorphism for this complex, whereas the COI gene is apparently uninformative.

Eudusbabekia choeronycteris new species was found on the phyllostomid bat, Choeronycteris mexicana Tschudi, in the central part of Mexico. The female, male, and larva are described and illustrated.

Bites attributed to the brown recluse spider, Loxosceles reclusa Gertsch & Mulaik, are frequently reported by medical personnel throughout Florida, whereas the extensive arachnological evidence contradicts the alleged widespread occurrence of Loxosceles spiders in the state. We compared reports of brown recluse spider bites made by medical personnel from a 6-yr Florida poison control center database to the known verifications of Loxosceles spiders from 100 yr of Florida arachnological data. Medical personnel diagnosed 124 brown recluse spider bites from 31 of Florida’s 67 counties in 6 yr. In contrast, only 11 finds of ≈70 Loxosceles spiders have been made in 10 Florida counties in 100 yr. Florida does not have sufficient widespread populations of Loxosceles spiders to warrant consideration of brown recluse spider envenomation as a probable etiology of dermonecrosis. Florida health care would improve if medical personnel would consider the multitude of other etiologies that manifest in dermonecrosis.

Reanalysis of counts of horn fly, Hematobia irritans (L.), obtained from a variety of cattle herds indicated that aggregation of the flies within herds decreased as mean fly density increased. Aggregation was also related to the proportion of fly-resistant and fly-susceptible cattle in a herd. Herds were grouped according to their degree of horn fly aggregation. Low aggregation herds included larger framed Angus, Horned Hereford, Polled Hereford, and Red Poll breeds. Moderate aggregation occurred with Brahman, Charolais, small-framed Angus, mixed cows, and Hereford × Charolais cross. High aggregation occurred with Chianina and mixed herds. Relationships between the sample means and variances varied among aggregation groups. A resampling approach was used to determine the influence of random sampling of a herd on the proportion of horn fly population estimates within fixed percentages of the true mean. The proportion of sample means within ± 5, 10, 15, and 20% of the true means varied with the proportion of the herd sampled, the mean and variance of fly density, and herd size. Recommendations for obtaining sample size to estimate fly density within a fixed percentage of the true mean are given.

The Anopheles quadrimaculatus s.l. (Say) complex consists of at least five species distinguished by distribution, genetic incompatibility, and allele frequencies. However, the distributions of the members have only been described by collection locations. Building on this information and environmental data, preliminary predictions of their distribution were produced using a genetic algorithm and point occurrence data. Based on resulting predicted border areas and undersampled regions, we obtained and analyzed additional geo-referenced specimens and compared their distribution with our preliminary predictions. We found good agreement between the preliminary predictions and the subsequent collections, regardless of the fact that additional specimens were deliberately sought from areas most likely to reveal inconsistencies. Final predicted distributions describe widespread distribution of A.quadrimaculatus throughout the eastern United States. A. maverlius and A. smaragdinus have similar predicted ranges limited to the southeastern United States. The predicted ranges of the sister taxa A. diluvialis and A. inundatus were similar to one another along the southeastern coast even though they seem to be allopatric. The historical role of A. quadrimaculatus s.l. in transmission of malaria was also examined. We conclude that A. quadrimaculatus s.s. was the only species of the complex capable of vectoring malaria in the United States throughout the area in which malaria occurred. However, any or all the members of the complex may have been regionally important, particularly in areas of most intense transmission.

An empirical model of flight initiation coupled with data from a longitudinal study predicted that the flight dispersal of Triatoma infestans from peridomestic sites was more likely to occur in late summer. To partially test this prediction, we operated 11–12 black light traps from 1945 to 2200 hours in March 2003 in two villages in northern Argentina. All peridomestic sites around the light traps were later inspected to assess the relative abundance and nutritional status of T. infestans at each site. Traps were located 19–94 m from the nearest infested site. A total of 2 female, 10 male, and 3 fifth-instar nymphs of T. infestans; 4 adult Triatoma garciabesi; and 1 Triatoma guasayana fifth-instar nymph were collected in 64 trap nights. Nearly two-thirds of the bugs arrived to the traps during the first hour after sunset, when ambient temperatures were 22–28°C; 80% of adults were unfed. The number of T. infestans that flew to the traps was significantly and negatively associated with wind speed, and the number of males positively associated with the abundance of adult T. infestans in peridomestic sites within 200 m around each light trap. This is the first successful application of light traps for collecting dispersing nymphal and adult T. infestans on a village-wide scale. We attribute this success to the placement of traps with consideration to spatial infestation patterns and seasonal variation in nutritional status of peridomestic triatomine populations.

Relationships between temperature and preoviposition, preeclosion, and premolt developmental periods for the tick Ixodes scapularis Say were investigated by holding field-collected ticks in the laboratory at temperatures of 0 to 32°C at constant daylength. The duration of these developmental periods decreased significantly with increasing temperature. Host of origin, prior storage at 4°C, and season of collection of the ticks were also significantly associated with variations in the duration of the preoviposition period. For each developmental stage, the effect of temperature on development rate was best described as a power relationship. Laboratory-derived relationships were used to predict dates for molting, oviposition, and eclosion of engorged larvae and nymphs, engorged adult females and egg masses, respectively, placed in the field during 1989–1992. Predicted dates for oviposition by adult females, eclosion of eggs, and molting of engorged larvae were within 2 wk of the observed dates, and field-observed seasonal activity of questing larvae and nymphs also was predicted well by laboratory data. Molting of engorged nymphs and seasonal activity of questing adult ticks were, however, poorly predicted. Our findings suggest that duration of development in the field, of larvae from engorged adult females, and of nymphs from engorged larvae, may be explained largely by temperature effects alone, whereas emergence of adult I. scapularis from engorged nymphs may depend on temperature-independent diapause phenomena. The significance of these findings for understanding current and future distributions of I. scapularis, and of the pathogens it transmits, is discussed.

Adult mosquito size may be related to longevity, feeding frequency, and other factors that impact vectorial capacity. Various investigations have shown that characteristics of larval mosquito habitats influence adult size. We studied breeding container characteristics in relation to size and abundance of Aedes aegypti larvae and pupae in Iquitos, Peru, and compared these with the size of resulting adult females. During 22 May to 20 July 2000, immature mosquitoes were collected from 12,722 containers in 2,931 houses, of which 424 held ≥1 Ae. aegypti. A subsample of larvae and all 16,433 pupae detected was removed for study. Resting adult mosquitoes were also collected from the same houses as the immatures. Adult mosquito size was determined by measuring the wing lengths of 672 aspirated adults and 2,316 adult females that emerged from container-derived pupae. Immatures were most commonly found in rain-filled containers, located outside of houses, and without lids. The average wing length of females derived from pupae varied considerably (1.67–3.83 mm), with slightly less variation for females captured as adults (1.80–3.23 mm). Linear regression showed that average wing length of pupae-derived females was positively associated with presence of larvae, container-filling method, diameter of container, and density of females. Size of pupae-derived females was correlated with that of females captured in the same houses as adults. The geographic distribution of pupae and adults indicated that the spatial pattern for Ae. aegypti is heterogeneous, with areas of higher and lower abundance. These findings provide insight for more focused control efforts aimed at reducing Ae. aegypti-borne pathogens.

Wing geometric morphometry was used to study the spatial structuring of populations of Triatoma infestans from different villages, ecotopes, and sites within a village in northwestern Argentina. A total of 308 male and 197 female wings of T. infestans collected from peridomestic and domestic ecotopes in March 2000 was analyzed. On average, female bugs had a significantly larger wing size than males. Triatomines collected from domiciles or structures associated with chickens had larger wings than bugs collected from goat or pig corrals. The wing size of bugs did not differ significantly between villages. Discriminant analyses of wing shape showed significant divergence between villages, ecotopes, and individual collection sites. The study of metric variation of males between sites belonging to the same ecotope also revealed significant heterogeneity. Indeed, within the same section of the village the difference between two goat corrals was sometimes greater than that between neighboring goat and pig corrals. Thus, morphometric heterogeneity within villages may be the result not only of ecotope and host associations, but also of physical isolation between subunits. The strong structuring of T. infestans populations in the study area indicates that recolonization could be traced back to a small geographic source.

Culex restuans Theobold, a putative vector of West Nile virus among birds in northern North America, also may serve as a bridge vector to mammals. Despite its potential public health importance, little is known about what regulates populations of this species. Mosquitoes generally are subject to both density-dependent reductions in survival and growth and to density-independent limitations on their population abundances. The mechanisms by which density dependence may occur in this species were examined in both field and laboratory studies. Nutrient-dependent reductions in growth were found in field studies. Under laboratory conditions, nutrient levels in larval habitats and total water volume per container contributed to survival and growth of larvae. We related these findings to density-independent changes in available habitat for larval development observed in other studies. These results may suggest a mechanism for patterns of mosquito abundance.

Isozyme frequencies in six wild-caught populations of Aedes albopictus (Skuse) from various parts of Thailand were compared using starch gel electrophoresis. Four populations were sampled from the south of the country, one from Samui Island and three from the mainland. The remaining two populations were obtained from central (Bangkok) and northern (Tak) Thailand. There were large differences in allele frequencies at two of 22 loci: Glutamate oxaloacetate transminase-2 (Got-2) and Hexokinase-1 (Hk-1). Got-2 (allele 100) was absent from the Bangkok population, whereas it was observed in high frequencies in all other populations. The Bangkok population showed the highest percentage of polymorphic loci (63.6%), whereas the population from Tak demonstrated the smallest percentage of polymorphic loci (18.2%).

Aedes aegypti is the main vector of dengue viruses responsible for dengue hemorrhagic fever, which has become a major public health concern in tropical countries. Because vaccines are still under development, dengue prevention depends entirely on vector control. Knowledge of gene dispersal patterns is required to develop efficient vector control strategies. Here we report the use of amplified fragment length polymorphism (AFLP) to infer the genetic structure of Ae. aegypti populations at a local levels (Phnom Penh, Cambodia). The amount of variation and patterns of gene flow detected are compared with those obtained with two other more widely used markers, isoenzymes and microsatellites. The pattern of differentiation depicted by AFLP data were confirmed by comparison of the F_st values of the three markers. Even though F_st values estimated with AFLP markers are three- to fivefold higher than those estimated with isoenzymes or microsatellites, these different markers reveal the same population structure. This technique is useful for population genetic studies of Ae. aegypti and is especially advantageous when few individuals specimens are available because of the ability to AFLP to simultaneously amplify large numbers of polymorphic DNA fragments.

Four fractions of Gloriosa superba L., i.e., hexane fraction, dichloromethane fraction 1, dichloromethane fraction 2, and methanol fraction, were investigated for colchicine-like activity using a mosquito cytogenetic assay. The results revealed that the latter three fractions yielded promisingly high colchicine-like activity, whereas the hexane fraction yielded very low activity compared with 1% colchicine in a 0.85% sodium chloride solution. The metaphase rates and average number of metaphase chromosomes per positive brain ganglion (range) of Aedes aegypti L. larvae after incubation with 0.25–2% solutions of dichloromethane fraction 1, dichloromethane fraction 2, 0.5–2% solutions of methanol fraction, and 1% colchicine solution were 90–100% and 7 (2–19) to 22 (7–47); 90–100% and 4 (1–11) to 30 (4–73); 95–100% and 11 (1–28) to 17 (2–62); and 100% and 6 (2–11), respectively. The temperature stability tests of the three promising fractions were performed by heating 0.5% working solution at 121°C for 15 min and preparing 0.5% working solution from stock frozen at −20°C for 10 mo. These fractions also yielded satisfactory outcomes of metaphase rates and an average number of metaphase chromosomes per positive brain ganglia compared with 1% colchicine solution.

Wolbachia are maternally inherited bacteria that infect a wide range of arthropods as well as filarial worms. The infection usually results in reproductive distortions of the host, primarily cytoplasmic incompatibility, parthenogenesis, and feminization. This study showed that Wolbachia infection (15/29; 51.72%) was prevalent among field-caught mosquitoes in Taiwan. Three mosquito species were identified as having Wolbachia A infection, eight species as having Wolbachia B, and four other species were dually infected by both groups. Each Wolbachia isolate from different mosquitoes was further divided into a specific subgroup. However, there were still some isolates that did not belong to any known subgroup, suggesting that more subgroups remain to be identified. Investigation of tissue tropism in either Aedes albopictus (Skuse) or Armigeres subalbatus (Coquillett) revealed that Wolbachia were extensively distributed within the host, although the ovary was most susceptible to infection. This report provides preliminary features of molecular relationships among Wolbachia groups of mosquitoes from Taiwan.

Dromyosuppressin (DMS) immunoreactive neurons were discovered running along the crop duct and on the surface of the crop in the house fly, Musca domestica L. DMS is a myoinhibitory neuropeptide that has been shown to inhibit crop contractions, in vitro, in the blow fly, Phormia regina (Meigen), and in Drosophila melanogaster Meigen. Various concentrations of benzethonium chloride (Bztc), an agonist of DMS with shown inhibitory effects on blow fly crop contractions, were fed to adult female M. domestica. Flies exhibited a dose-dependent mortality; avoidance and subsequent dehydration are probably the cause of the low survivorship at higher Bztc concentrations.

Anopheles stephensi (Liston) is a well-known vector of malarial parasite in tropical countries. The developing trend of resistance in mosquitoes toward synthetic mosquitocidal agents makes their management extremely difficult. Effectiveness of essential oils with aroma therapeutic values seems to be an emerging tool to combat this vector. Piperitenone oxide isolated from essential oil of a new genotype, Mentha spicata L. variety viridis, has been evaluated for larvicidal, ovicidal, oviposition-deterrent, developmental toxicity, and repellent properties against various stages of A. stephensi. The results indicated the higher efficacy of piperitenone oxide than the crude essential oil of M. spicata variety viridis in all the bioassay experiments. The lethal response of piperitenone oxide and the oil toward fourth instar larvae showed LD₅₀ values of 61.64 and 82.95 μg/ml, respectively. Female adults of A. stephensi exposed to the oil laid ≈42 times less number of eggs at the dose of 60.0 μg/ml as compared with control, whereas exposure of piperitenone oxide at the same dose completely inhibited the oviposition. Furthermore, piperitenone oxide also completely inhibited egg hatching at the dose of 75.0 μg/ml in ovicidal assay. Developmental toxicity studies showed the significant developmental inhibition potential of the compound and oil. Additionally, piperitenone oxide was found to be highly toxic and repellent toward adults of A. stephensi as compared with oil.

The insecticidal activity of 54 plant essential oils against female Pediculus humanus capitis De Geer was examined using direct contact and fumigation methods, and compared with the lethal activity of δ-phenothrin and pyrethrum, two commonly used pediculicides. In a filter paper contact bioassay with female P. humanus capitis, the pediculicidal activity was more pronounced in eucalyptus, marjoram, pennyroyal, and rosemary oils than in δ-phenothrin and pyrethrum on the basis of LT₅₀ values at 0.0625 mg/cm². At 0.125 mg/cm², pediculicidal activity of cade, cardamone ceylon, clove bud, myrtle, rosewood, and sage oils was comparable with that of the test insecticides. In fumigation tests with female P. humanus capitis at 0.25 mg/cm², eucalyptus, marjoram, pennyroyal, and rosemary oils were more effective in closed containers than in open ones, indicating that the effect of these oils was largely a result of action in the vapor phase. Neither δ-phenothrin nor pyrethrum exhibited fumigant toxicity. The essential oils described herein merit further study as potential control agents for P. humanus capitis.

Nymphal and adult ticks from three different tick species, Dermacentor variabilis Say, Ixodes scapularis Say, and Rhipicephalus sanguineus Latrielle, were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae Metschnikoff. Dose-response experiments indicated that a critical concentration of fungal spores is required for infection and mortality. Over a 28-d time course, fungal suspensions of either B. bassiana or M. anisopliae at 10⁸ conidia/ml resulted in 50–70% mortality in adult I. scapularis and R. sanguineus, but <20% mortality in D. variabilis ticks. R. sanguineus nymphs were highly susceptible to both entomopathogenic fungi, displaying >60% mortality within 14 d postinfection and >90% mortality within 21–28 d postinfection. D. variabilis nymphs also were more susceptible than their corresponding adults, displaying mortalities ranging from 20 to 40% 28 d postinfection. I. scapularis nymphs, however, seemed to be slightly less susceptible than adults (45% mortality, 28 d postinfection). The addition of nutrients to fungal cell suspensions did not have any noticeable effects on mortality toward any of the tick species tested. Significant mortality against D. variabilis adults (≈65%) was noted only when B. bassiana fungal cells with growth media carryover were used as the inoculum against the ticks. Entomopathogenic fungi such as B. bassiana and M. anisopliae may have the potential for controlling populations of I. scapularis and R. sanguineus, and under certain conditions D. variabilis. Our results indicate that inoculum conditions can greatly affect successful virulence and subsequent mortality.

Because pyrethroid, organophosphate, and carbamate resistance is more and more developed in mosquitoes of medical importance, there is an urgent need for alternative insecticides for vector control. Dinotefuran, a new neonicotinoid insecticide commercialized by Mitsui Chemicals (Tokyo, Japan), could be a useful candidate in public health because it shows low mammalian toxicity and great insecticidal activity against a broad range of pests. In this study, the intrinsic toxicity of dinotefuran was evaluated by larval bioassay and topical application against different mosquito strains of Anopheles gambiae Giles, Culex quinquefasciatus Say, and Aedes aegypti L. having none, one, or several resistance mechanisms, respectively, to insecticides. The results showed that dinotefuran was less toxic than most of the commonly used insecticides (e.g., deltamethrin, carbosulfan, and temephos) against the susceptible mosquitoes tested (between 6- and 100-fold at the LD₅₀ level). However, the toxicity of dinotefuran was not strongly affected by the presence of common resistance mechanism, i.e., kdr mutation and insensitive acetylcholinesterase (resistance ratio [RR] from 1.3 to 2.3). More interestingly, the carbamate-resistant strain of Cx. quinquefasciatus was significantly more affected by dinotefuran than the susceptible strain (RR = 0.70), probably because the insensitive acetylcholinesterase is less efficient to degrade nicotinic substrates than normal acetylcholinesterase. Despite the relatively low toxicity of dinotefuran against susceptible mosquitoes, the absence of cross-resistance with common insecticides (pyrethroids, carbamates, and organophosphates) makes neonicotinoids potential candidates for disease vector control, especially in area where mosquitoes are resistant to insecticides.

Population surveys of Culex pipiens quinquefasciatus carried out in 1991 and 1999 were compared with data collected in 1990 before the beginning of the control program against this mosquito. Larval samples collected in 1999 displayed resistance to the four tested insecticides: permethrin, propoxur, temephos, and chlorpyrifos. Temephos resistance ratio at LC₅₀ (RR₅₀) ranged between 8.1- and 42-fold compared with 2.9- and 4.6-fold in 1990, and chlorpyrifos RR₅₀ ranged between 8.6- and 123-fold compared with 6.4- and 19-fold in 1990. This increased resistance to organophosphorus insecticides was associated with a sharp decrease of susceptible genotypes at two loci (Ester and ace-1), as well as to an allele replacement at the Ester locus.

Four synthetic mosquito repellents (Autan [10% KBR3023], IR3535 [7.5%], Off! [15% deet], Skinsations [7% deet]) and eight natural (primarily plant extracts and/or essential oils) product-based repellents (Bite Blocker [2% soybean oil], ByGone, GonE!, Natrapel [10% citronella], Neem Aura, Sunswat, MosquitoSafe [25% geraniol], and Repel [26% p-menthane-3,8-diol]) were tested in the laboratory against Aedes albopictus Skuse, Culex nigripalpus Theobald, and Ochlerotatus triseriatus (Say). When estimated mean protection time (eMPT) responses for each repellent were averaged for all three mosquito species, Autan, Bite Blocker, Off!, and Repel prevented biting for ≥7.2 h; IR3535, MosquitoSafe, and Skinsations for 3.2–4.8 h; and ByGone, Natrapel, GonE, NeemAura, and SunSwat for 0.9–2.3 h. Against Ae. albopictus, the eMPT for Off! and Repel exceeded 7.0 h and ranged from 5.0 to 5.7 h for Autan, Bite Blocker, and Skinsations. Bygone, GonE, NeemAura, and SunSwat provided 0.2 h protection against Ae. albopictus and Oc. triseriatus, whereas Autan, Bite Blocker, Off!, and Repel prevented bites by Oc. triseriatus for ≥7.3 h. All 12 repellents provided an eMPT ≥2.8 h against Cx. nigripalpus (maximum: 8.5 h for Bite Blocker). When the average eMPT for each repellent (for all species) was divided by the eMPT for 7% deet (Skinsations), the order of repellent effectiveness and the corresponding repellency index (R_i) was Repel (1.7) > Bite Blocker (1.5) = Autan (1.5) = Off! (1.5) > Skinsations (1.0) > IR3535 (0.8) > MosquitoSafe (0.6) > Natrapel (0.5) > Neem Aura (0.3) = SunSwat (0.3) = Bygone (0.3) > GonE (0.2).

In a West Nile virus (WNV)-free ecosystem, it is essential to verify the integrity of RNA before concluding that RNA extracted from mosquito specimens is negative for WNV gene sequences. The primary objective of our study was to develop a rapid molecular assay to rapidly screen mosquitoes for the presence of 18S RNA and WNV gene sequences. Mosquitoes, collected from multiple sites on the island of O‘ahu, were pooled into groups of 1–50 mosquitoes according to capture site, date, and species. Using primer design software and the GenBank database, generic oligonucleotide primer pairs were designed to amplify mosquito18S rRNA gene sequences from different species. RNA was extracted from mosquito pools, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for the presence of mosquito18S rRNA and WNV gene sequences. Three of the seven primer pairs successfully detected 18S rRNA sequences for both Aedes and Culex by RT-PCR, and one primer pair successfully amplified 18S rRNA sequences for 15 different mosquito species. All 64 mosquito pools from 10 different sites on the island of Oahu, Hawaii, were negative for WNV nonstructural protein-5 gene sequences. This simple, one-step RT-PCR method for screening mosquito pools for arboviruses will become an increasingly valuable tool as WNV becomes endemic throughout the Americas.

This is the first report of Borrelia burgdorferi sensu lato in rodents from San Luis Obispo county, with most isolates obtained from a previously unreported host, Neotoma lepida Thomas. B. burgdorferi sensu lato was identified in seven rodent species, including the California vole, Microtus californicus Peale; dusky-footed woodrat, Neotoma fuscipes Baird; desert woodrat, Neotoma lepida Thomas; brush mouse, Peromyscus boylii Baird; California mouse, Peromyscus californicus Gambel; deer mouse, Peromyscus maniculatus Wagner; and western harvest mouse, Reithrodontomys megalotis Baird. Ear punch biopsies were cultured in BSK-H medium from 179 rodents trapped at six different study sites. Overall, prevalence of rodent infection was 44/179 (24.6%), with 34 of these isolates from N. lepida. Spirochete isolates were obtained from rodents at all study sites, indicating widespread prevalence of B. burgdorferi sensu lato across rodent species and habitats. Nucleotide sequences for 14 of these isolates have been submitted to GenBank. Isolates from three N. lepida and one P. boylii had identical flagellin gene sequences, and phylogenetic analysis placed these spirochetes in B. burgdorferi sensu lato group DN127, now known as B. bissettii Postic, Marti Ras, Lane, Hendson & Baranton. Additional sequencing of the intergenic spacer regions between the 5S and 23S ribosomal genes was performed on three of these isolates. Phylogenetic analysis separated these isolates into two clusters that grouped with Colorado or California isolates. The role of B. bissettii and related species other than B. burgdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt & Brenner as human pathogens in the United States warrants further investigation.

Environmental risk factors associated with increased malaria mosquito (Anopheles) abundance indoors were studied in a suburban area, Antula, of Guinea Bissau, during the rainy seasons of 1993–1995. All bedrooms in 30 houses were searched for resting mosquitoes three times each year. The most abundant mosquito was An. gambiae s.s. Significantly greater numbers of resting mosquitoes were present in rooms with open eaves and in houses with a well on the compound. Pigs were the most common domestic animals in Antula. Presence of pigs in a house was associated with increased mosquito abundance in the bedrooms of the same house. The abundance of female mosquitoes also increased with increasing human biomass per square meter of bedroom area.

Amblyomma americanum (lone star tick) is known or suspected to vector several organisms that are implicated as human pathogens, including Ehrlichia chaffeensis, E. ewingii, and Borrelia lonestari. These three agents have also been detected in white-tailed deer (Odocoileus virginianus). Because northeastern Georgia has a high abundance of both lone star ticks and white-tailed deer, and one of these organisms, E. chaffeensis, is already known to be endemic in the area, we assayed individual adult A. americanum, collected during the spring of 2001, 2002, and 2003, for these three organisms. A total of 400 ticks were dissected and tissues assayed by polymerase chain reaction (PCR) using Ehrlichia species-specific and Borrelia genus-wide primers. Of ticks tested, 2.0% (8/398) had evidence of E. chaffeensis, 4.8% (19/398) had evidence of E. ewingii, and 1.0% (4/398) had evidence of B. lonestari. Borrelia sp. spirochetes were also visualized by an indirect fluorescent antibody test, using an anti-flagellin monoclonal antibody (H9724), in a total of 10.7% (32/300) of ticks tested in 2003. These results reconfirm the presence of E. chaffeensis and establish evidence of E. ewingii and B. lonestari in questing adult A. americanum ticks from northeastern Georgia. Detection of at least two of the three organisms in ticks collected each year suggests that people in northeastern Georgia are at risk of infection with these organisms.

Control of Chagas disease requires control of its triatomine vectors, which requires an understanding of the determinants of infestation. Twenty-seven household environmental characteristics in the town of Chalcatzingo, Morelos, were analyzed for association with infestation by Triatoma pallidipennis, the predominant local vector. Data were obtained through timed household searches for triatomines and surveys that characterized intradomicile and peridomicile environments. Of the households surveyed, 28.4% were infested by T. pallidipennis. Cross-sectional multivariate logistic regression analyses were performed that regressed infestation on environmental variables. Of the 530 households in the town, 84% had sufficient data to be included. Adobe walls, agricultural products, junk piles, lack of bednets, and number of rabbits were significantly associated with intradomiciliary infestation. Junk piles and numbers of dogs, cats, and rabbits were significantly associated with peridomiciliary infestation. Junk piles, agricultural products, and numbers of cats, rabbits, and birds were significantly associated with overall infestation. Unexpectedly, presence of stone piles was not associated with infestation. The results of this study provide information for designing Chagas disease control programs in rural Mexican areas infested by T. pallidipennis.

The primary aims of this study were to quantify the density of Ixodes pacificus Cooley and Kohls nymphs and adults of the same generational cohort collected within a single year in six oak or madrone leaf litter habitats and to compare the prevalence of infection with Borrelia burgdorferi sensu lato (s.l.) in adults originating from nymphal cohorts with a low (<1%) versus high (>10%) infection prevalence. Because adult densities were very low both in and adjacent to several sites, direct comparisons of infection prevalence between nymphs and adults were possible only for two sites. Mean density in these sites decreased from 11.95/100 m² for nymphs to 0/100 m² for adults in leaf litter, and infection prevalence with B. burgdorferi s.l. was four-fold higher in nymphs (7.4%) versus adults (1.6%) of the same generational cohort collected in ecotones bordering the leaf litter areas. Assuming a density of adults in leaf litter of 0.04/100 m² (mean for all six examined sites) and an infection prevalence similar to that found in adults collected from litter ecotones, the risk of encountering infected ticks in leaf litter decreased >1,000-fold from the nymphal to adult stage. Regardless of site-specific infection prevalence in the nymphal stage (n = 2 sites; 0.7 versus 14%), the infection prevalence for the adults of the same generational cohort was similarly low (1.5–1.6%). Peak densities of adult I. pacificus were 0–0.1/100 m² in leaf litter, 0–6.5/100 m² in ecotonal grasslands, and 2.0–39.0/100 m² in ecotonal chaparral. Despite more intensive sampling efforts in leaf litter, the vast majority of the 282 adults collected came from grass or chaparral ecotones (98.9%, n = 279) rather than leaf litter (1.1%, n = 3). The study yielded eight B. burgdorferi s.l.–infected adults; four of these carried B. burgdorferi sensu stricto Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, and the remaining four were infected with currently undescribed B. burgdorferi s.l. spirochetes. This is the first study comparing both density of I. pacificus nymphs and adults and prevalence of infection with B. burdorferi s.l in these ticks within the same generational cohort and sampling area.

Visceral leishmaniasis is an endemic protozoal disease of humans and dogs in tropical and subtropical regions in Asia, Africa, southern Europe, Central America, and South America, where sand flies (genera Phlebotomus and Lutzomyia) act as vectors. An outbreak in a New York foxhound kennel and subsequent surveillance revealed widespread Leishmania infantum infection of dogs in the United States, outside the known range of the vector sand flies. For this study, we conducted surveillance for sand flies during the summers of 2001 and 2002 at two areas: on the grounds of the New York kennel and at the Institute of Ecosystem Studies (IES) 10 km away. CO₂-baited light traps were used for surveillance. Populations of Lutzomyia vexator, not previously known in New York, were widespread and locally abundant (range, 0.26–1.16 flies/trap night) at the IES site. These populations showed a bimodal, midsummer activity peak and were most abundant on steep slopes within mature mixed hardwood forests. Further research will be necessary to determine whether the New York populations of L. vexator in the vicinity of the kennel could be involved in transmission of canine leishmaniasis.

Monhegan is an isolated 237-ha island lying 16 km off the coast of Maine. Introduced to the island in 1955, white-tailed deer, Odocoileus virginianus Zimmerman, reached a density of ∼37/km² by the mid-1990s. Black-legged ticks, Ixodes scapularis Say, first noticed in the late 1980s, flourished thereafter. Norway rats (Rattus norvegicus Berkenhout) on Monhegan are highly infected with Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, the agent of Lyme disease. By 1996, 13% of year-round residents had contracted the disease. The community’s subsequent decision to eliminate deer from the island provided a unique opportunity to monitor the abundance of vector ticks in response to the complete and permanent removal of the primary hosts of their reproductive stage. With the exception of humans and their dogs and cats, there are no other potential hosts for adult I. scapularis on Monhegan. From November 1996 to March 1999, all deer were removed from the island. Previous annual fall flagging of vegetation from 1990 to 1998 produced 6–17 adult ticks/h, of which 24–41% were infected with the Lyme disease spirochete. During this same period, up to 18 larvae and 4 nymphs were removed per Norway rat live-trapped on the island each July. With the absence of deer in the fall of 1999, both the density of host-seeking adult ticks and infection prevalence rose substantially to 28/h and 75.0%, respectively. By the summer of 2003, however, no sub-adult ticks were found on rats, and that fall, only 0.67 adult ticks/h were flagged. Of the 68 adults collected from 2002 to 2003, 20 (29.4%) were infected. Over this same period, adult tick abundance on a deer-populated, reference island continued to gradually increase.

Studies of carrion-insect succession on domestic pig, Sus scrofa L., were conducted in the spring and summer of 2001 and 2002 in Blacksburg, VA, to identify and analyze the successional patterns of the taxa of forensic importance in southwest Virginia. Forty-seven insect taxa were collected in the spring. These were represented by 11 families (Diptera: Calliphoridae, Sarcophagidae, Muscidae, Sepsidae, Piophilidae; Coleoptera: Staphylinidae, Silphidae, Cleridae, Trogidae, Dermestidae, Histeridae). In the summer, 33 taxa were collected that were represented by all of the families collected in the spring, except Trogidae. The most common flies collected were the calliphorids: Phormia regina (Meigen) and Phaenicia coeruleiviridis (Macquart). The most common beetles were Creophilus maxillosus L. (Staphylinidae), Oiceoptoma noveboracense Forster, Necrophila americana L., Necrodes surinamensis (F.) (Silphidae), Euspilotus assimilis (Paykull), and Hister abbreviatus F. (Histeridae). Occurrence matrices were constructed for the successional patterns of insect taxa during 21 sampling intervals in the spring and 8 intervals in the summer studies. Jackknife estimates (mean ± 95% confidence limits) of overall Jaccard similarity in insect taxa among sampling intervals in the occurrence matrices were 0.213 ± 0.081 (spring 2001), 0.194 ± 0.043 (summer 2001), 0.257 ± 0.068 (spring 2002), and 0.274 ± 0.172 (summer 2002). Permutation analyses of the occurrence matrices showed that the patterns of succession of insect taxa were similar between spring 2001 and 2002 (P = 0.001) and between summer 2001 and 2002 (P = 0.007). The successional patterns seem to be typical for the seasonal periods and provide data on baseline fauna for estimating postmortem interval in cases of human death. This study is the first of its kind for southwest Virginia.

We demonstrated in this study that blood group O subjects attracted more Aedes albopictus than other blood groups (B, AB, and A) but were only significantly more attractive than blood group A subjects in 64 human landing tests. We collected saliva from the subjects and tested it for agglutination inhibition, categorized the subjects into secretors or nonsecretors, and studied mosquitoes’ landing preferences for those groups. The mean relative percent landing on blood group O secretors (83.3%) was significantly higher than on group A secretors (46.5%). We also compared the attraction to subjects according to blood groups using forearm skin treated with ABH antigens. Blood group O disaccharide (H antigen) attracted significantly more Ae. albopictus than did blood group A trisaccharide (A antigen), and subjects treated with blood group A disaccharide attracted significantly more Ae. albopictus than did subjects treated with blood group B trisaccharide (B antigen), but ABH antigens did not, in general, influence the landing preference of mosquitoes among ABO blood groups.

The first technique for repeatedly sampling individual Ixodes scapularis adult ticks was developed and validated. Gut samples from the same individual ticks were removed and analyzed at weekly intervals. Multiple analyses were conducted on each gut sample (e.g., total protein concentration, presence of viable B. burgdorferi spirochetes, and concentration of outer surface protein A [OspA]). Female I. scapularis survived for up to 25 d after gut sampling. Seventy-five percent of females oviposited after the sampling procedure, with 14% of ticks laying >1,500 eggs. No significant differences in either fecundity or length of survival were found between B. burgdorferi–infected and uninfected I. scapularis. This technique will enable longitudinal studies on both tick–pathogen interactions and physiological studies that have hitherto not been attempted.

Hematophagus arthropod bloodmeals may be useful in identifying individual hosts. To examine the application of human lice as a forensic tool, that is, as evidence of physical contact between individuals, body lice from a laboratory colony and head lice, collected from the head of infested children, were studied. The DNA profile of an individual was detectable in the pooled bloodmeals of two body lice, up to 20 h postfeeding. A mixed DNA profile of two hosts was identifiable in the pooled bloodmeals of three lice, for 3 h postfeeding. By pooling the bloodmeals of three adult and three nymphal head lice, a mixed DNA profile was obtained. These results indicate that, in criminal cases where there has been close contact between assailant and victim, louse bloodmeals should not be overlooked as potentially critical evidence.

Temporal changes in the cuticular hydrocarbons of female Anopheles stephensi (Liston) (Diptera: Culicidae) were quantified using gas-liquid chromatography with flame-ionization detection. The ratio of two prominent hydrocarbons, nonacosane (C₂₉) and hentriacontane (C₃₁), was found to change significantly with respect to mosquito age over a period of 15 d. A regression model was developed using this ratio, C₂₉/C₃₁ = 3.96 − 1.63 log (age), and prediction intervals, based on a 12-d developmental interval necessary for females to transmit malaria, were generated using confidence levels for one-sided tests. The model predicted that females that had a C₂₉/C₃₁ ratio of 2.6 or greater were only 10% probable to be old enough to transmit malaria, whereas females with ratios of 1.8 or less were 90% probable.

Anopheles gambiae Giles larvae usually live near the surface of shallow and temporary aquatic habitats. How deep the larvae can dive and how long they can submerge may be related to feeding efficiency and predator avoidance. This study examined diving behavior of An. gambiae larvae in the laboratory. We recorded diving depths and larval mortality of second and fourth instars in clean water and muddy water by using deep water (32-cm) and shallow water (20-cm) columns. In deep water columns with clean water, we found that 2% of second instars and 6% of fourth instars died from diving, whereas 3% of second instars and 11% of fourth instars died in muddy water. The fourth instars dived deeper in muddy water than in clean water. The mortality rates of the fourth instars subjected to diving stimulations were significantly higher than those in the shallow water columns. Therefore, larval diving behavior may offer the benefits of predator avoidance and food acquisition but also incur energetic costs and increased mortality.