Understanding the ecology and behavior of different mosquito species (Diptera: Culicidae) is essential for identifying their role in disease transmission cycles and public health risk. Two species of Culex mosquitoes in the northeastern United States, Culex pipiens L. and Culex restuans Theobald, have been implicated in enzootic transmission of West Nile virus (family Flaviviridae, genus Flavivirus, WNV). Despite the difficulty of differentiating these two species as adults, many public health workers and vector biologists collecting adults in the field separate these species based on external morphology. This approach is often used rather than examination of dissected male genitalia or polymerase chain reaction (PCR)-based diagnostics due to time or cost constraints. We evaluated the reliability of seven published morphological characters to differentiate adults of these species by comparing blindly scored morphology with PCR-based confirmations. Our study demonstrates that morphological identification of Cx. pipiens is marginal and often not reliable for Cx. restuans. We also examined error rates with molecular-based approaches. DNA samples were contaminated with as little as one leg from another species. We conclude that to fully understand the respective roles of Culex species in the epidemiology of WNV and other pathogens, more attention should be paid to these considerations for accurate species identification.

An in vitro feeding method using chicken-skin membranes and human blood was compared with an established in vivo method using anesthetized hamsters for blood-feeding mass-reared phlebotomine sand flies. Parameters measured were percentage of sand flies taking blood meals, number of eggs laid per female, and percentage of eggs that hatched. Females from a long established (>20 yr) colony of Phlebotomus papatasi (Scopoli) from Israel landed on and started feeding sooner on the hamster than on the membrane. However, when sand flies were allowed access to the membrane feeder for the same length of time as the anesthetized hamster, the feeding percentages were not significantly different and were usually better on the membrane feeder if flies were allowed access for a longer time. Egg production and percent hatch between the two feeding methods were not statistically different. Based on these results, we conclude that the chicken-skin membrane feeding method is a viable alternative to the use of live animals for feeding large numbers of P. papatasi.

Triatoma pseudomaculata Corrêa and Espínola, 1964 and Triatoma juazeirensis Costa and Felix, 2007 (=T. brasiliensis Neiva, 1911 [part]), are sylvatic vectors of Trypanosoma cruzi (Chagas, 1909), the causative agent of Chagas disease, in northeast Brazil—especially in the caatinga region. In an area of caatinga in the State of Bahia, we compared the wild and peridomestic habitats of these two species of Triatominae to assess their behavioral plasticity in relation to habitat selection in different environments. In the sylvatic environment, the habitat of these two species is never shared. T. pseudomaculata is found in trees and bird nests, but without apparent preference for any particular tree species. In contrast, T. juazeirensis is exclusively rupicolous (found among rocks). Both species invade peridomestic structures but do not display a significant ability to colonize human dwellings. In the peridomestic area, they are highly adaptable to different habitats and can occupy substrates that they do not colonize in the sylvatic environment. This behavioral plasticity seems to be more striking in T. juazeirensis—rupicolous in sylvatic environments but colonizing wooden structures in the peridomicile in >80% of cases.

Many invasive species succeed in becoming established in new locations because of their competitive superiority to native species. This has been shown in several examples involving mosquitoes. In this study, we examined the interspecific competition between mosquito larvae of a well-established, non-native species, Culex pipiens, and those of its ecologically similar, native congener Culex restuans. Small but significant differences in survival, growth, and development rates were found in Cx. restuans as a response to varying proportions of Cx. pipiens, suggesting that Cx. restuans is a slightly superior competitor. However, the overall differences between the species were small, and they may be nearly ecological equivalents as larvae. Nevertheless, the observed seasonal pattern of feeding and oviposition activity suggests some phenological avoidance of competition, thus demanding further study of the interaction of these two species.

We investigated the role of heavy rain on container-inhabiting mosquito (Diptera: Culicidae) populations, and how different species may have adapted to such conditions. Rains were created with a rain simulator calibrated to natural rain intensities in the habitats of two important vector species: Aedes aegypti (L.) from northern Thailand and Culex pipiens L. from New York state, USA. Immature stages of Ae. aegypti were able to resist the flushing effect of rain better than Cx. pipiens. This difference was most dramatic during the pupal stage. Fourth instars of Ae. aegypti were not affected by flushing when exposed for longer rain intervals (30 versus 60 min) or at a colder water temperature (24 versus 16°C). In contrast, significantly more Cx. pipiens larvae flushed out with longer rain exposure. Warmer water temperatures also increased the proportion of Cx. pipiens flushed out, but mostly at the longest exposure time. Container position (tilted at a 7° angle or level) did not affect proportions of fourth instars flushed out for both species. More accurate models of vector-borne diseases can be developed by incorporating the described effects of rain on container-breeding mosquito populations. Such models may provide more realistic assessments of disease risk and ensure optimal use of limited financial resources of mosquito control agencies.

A variety of refugia were offered to different instars of brown recluse spiders, Loxosceles reclusa Gertsch and Mulaik, and a South American recluse spider, L. laeta (Nicolet), to determine whether they preferred certain types of refugia spaces. Variables included (1) crevice widths ranging from 3.2 to 21 mm, (2) horizontal and vertical orientations, and (3) new refugia or refugia that had silk deposited by a previous conspecific resident. An additional 30-d assay with similar-sized refugia studied each species’ propensity for site fidelity or movement among refugia. L. reclusa preferred crevice widths ≥9 mm with no correlation of body size to crevice width, whereas L. laeta preferred crevice sizes ≥6.4 mm with a marginally significant correlation between crevice width and body size. Both species preferred (1) vertical instead of horizontal-oriented refugia and (2) refugia with conspecific silk compared with previously uninhabited refugia. There was no significant difference between the species in their propensity to move among refugia in the 30-d trial; however, both species had individuals that were always found in the same refugium for the entire assay and individuals changing refugia every 2–3 d. The propensity to switch refugia was not affected by the degree of starvation for the period tested as was initially hypothesized. The possible implications of this research toward developing novel control measures for Loxosceles spiders are discussed.

A survey (2005–2006) of house fly, Musca domestica L. (Diptera: Muscidae) populations on four Florida dairy farms demonstrated the presence of flies with acute symptoms of infection with salivary gland hypertrophy (SGH) virus on all farms. Disease incidence varied among farms (farm averages, 0.5–10.1%) throughout the year, and it showed a strong positive correlation with fly density. Infections were most common among flies that were collected in a feed barn on one of the farms, especially among flies feeding on wet brewers grains (maximum 34% SGH). No infections were observed among adult flies reared from larvae collected on the farms, nor among adults reared from larvae that had fed on macerated salivary glands from infected flies. Infected female flies produced either no or small numbers of progeny, none of which displayed SGH when they emerged as adults. Healthy flies became infected after they fed on solid food (a mixture of powdered milk, egg, and sugar) that had been contaminated by infected flies (42%) or after they were held in cages that had previously housed infected flies (38.6%). Healthy flies also became infected after they fed on samples of brewers grains (6.8%) or calf feed (2%) that were collected from areas of high fly visitation on the farms. Infection rates of field-collected flies increased from 6 to 40% when they fed exclusively on air-dried cloth strips soaked in a suspension of powdered egg and whole milk. Rates of virus deposition by infected flies on food were estimated by quantitative polymerase chain reaction at ≈100 million virus copies per fly per hour. Electron microscopy revealed the presence on enveloped virus particles in the lumen of salivary glands and on the external mouthparts of infected flies.

Seven Triatoma dimidiata (Latreille, 1811) populations from different provinces of Guatemala were compared along with three related triatomine species using the electrophoretic profiles of salivary proteins. The analysis of salivary proteins allowed the separation of two of the species into their respective complexes, phyllosoma (T. pallidipennis) and protracta (T. nitida) (Lent and Wygodzinsky, 1979), whereas T. dimidiata seems slightly separated from either of these. Based on salivary protein profiles, T. dimidiata is most closely related to the cluster including T. ryckmani and T. nitida (protracta) and more diverged from T. pallidipennis (phyllosoma). Among Guatemalan T. dimidiata populations, the cave population from Lanquin is separated from the rest of populations analyzed, suggesting that it is in the process of speciation. No difference in protein banding pattern was observed among populations from domestic and peridomestic ecotopes from the same region.

The tropical mosquito Aedes aegypti (Diptera: Culicidae) is the most important domestic vector of urban yellow fever and dengue viruses. Ae. aegypti originated from Africa and was probably introduced into Brazil during the colonial period through embarkations, and dengue epidemics soon followed. Genetic analysis of 12 Ae. aegypti populations from five states in Brazil was conducted based on two mitochondrial DNA fragments: cytochrome oxidase I and NADH dehydrogenase subunit 4. Analyses comparing individual haplotypes indicated the existence of two well-defined clades, probably representing two mitochondrial lineages. Analysis of molecular variance showed significant variability in genetic structure among collections within groups. Mantel regression analysis showed a correlation between genetic and geographic distances, mainly because of northern and northeastern populations, in comparison with those in the southeast. The population from Santos, the largest port in Brazil, showed the greatest diversity, with 10 unique haplotypes, an indication of recent introductions that have not yet spread to other Brazilian cities. Different mitochondrial DNA sequences were found in three specimens, indicating the presence of heteroplasmy.

A cDNA encoding a putative aquaporin was cloned from the ovaries of the American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae). The encoded protein is most similar to the vertebrate aquaporin 9 protein family. Localization by reverse transcription-polymerase chain reaction (RT-PCR) shows expression in the gut and ovaries of adult females but not in the synganglion, Malpighian tubules, or salivary glands. Quantitative RT-PCR indicates that it is primarily expressed in the ovaries, with ≈146 times more transcript than in the gut. When expressed in Xenopus oocytes, the aquaporin-like protein localized to the plasma membrane.

This study examines the effectiveness of a new ivermectin formulation for the topical treatment of the human head louse, Pediculus humanus capitis De Geer (Anoplura: Pediculidae). Permethrin-resistant lice originally obtained from south Florida and maintained on an in vitro rearing system were 100% susceptible to ivermectin formulations by using a semiclinical hair tuft bioassay. The formulation was 100% effective at killing lice using 1, 0.5, and 0.25% ivermectin concentrations after 10-min exposures. As judged by the lethal time (LT)₅₀ and LT₉₅ values, 0.5% formulated ivermectin was 3.8 and 3.2 times faster at killing lice, respectively, than 0.5% nonformulated ivermectin, indicating that the formulation may facilitate the penetration of ivermectin into the louse. The hair tuft-based bioassay in conjunction with the in vitro rearing system provides a standardized method to assess the comparative efficacy of pediculicide formulations in a reproducible format that mimics the exposure scenario that occurs on the human scalp.

To access the relative potency of pesticides to control adult mosquitoes, 19 pesticides with various modes of action were evaluated against Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say. On the basis of 24-h LD₅₀ values after topical application, the only pesticide that had higher activity than permethrin was fipronil, with LD₅₀ values lower than permethrin for 107-, 4,849-, and 2-fold against Ae. aegypti, Cx. quinquefasciatus Say, and An. quadrimaculatus Say, respectively. Abamectin, imidacloprid, spinosad, diazinon, and carbaryl showed slightly lower activity than permethrin (<20-fold). However, bifenazate showed very low activity against the three mosquito species tested, with LD₅₀ values higher than permethrin for >1000-fold. On the basis of 24-h LD₅₀ values, Cx. quinquefasciatus was the least susceptible species to nine pesticides tested (DNOC, azocyclotin, chlorfenapyr, carbaryl, spinosad, imidacloprid, diazinon, abamectin, and permethrin), whereas Ae. aegypti was the least susceptible species to six pesticides tested (dicofol, amitraz, propargite, hydramethylnon, cyhexatin, and diafenthiuron), and An. quadrimaculatus was the least susceptible species to four pesticides tested (bifenazate, pyridaben, indoxacarb, and fipronil). Our results revealed that different species of mosquitoes had different susceptibility to pesticides, showing the need to select the most efficacious compounds for the least susceptible mosquito species to achieve successful mosquito control.

The repellent effect of the essential oils of flower heads of the aromatic plant tansy, Tanacetum vulgare L. (Asteraceae), originating from Sweden, was tested against host-seeking nymphs of the common tick Ixodes ricinus (L.). The essential oils were obtained by steam distillation (SD) and by using an online solvent extraction separation setup. Further fractionations of the SD oils were obtained by medium-pressure liquid chromatography on silica gel. The volatiles of the essential oils and the fractions that exhibited strong tick repellency (90–100%) were collected by solid phase microextraction and identified by gas chromatography-mass spectrometry. The chemical analyses of the oils show that the populations of T. vulgare from Uppsala and Stockholm may represent different chemotypes, but that they exhibited similar tick repellency. Main volatiles detected from oils of T. vulgare collected at Uppsala were α-pinene (27%), β-pinene (11%), pinocamphone (11%), 1,3,3-trimethylcyclohex-1-ene-4-carboxaldehyde (11%), and 1,8-cineole (10%). In the sample collected in Stockholm, the main components were β-thujone (39%) and camphor (23%) followed by α-thujone (11%) and 1,8-cineole (8%). When constituents in the oils, e.g., α-terpineol, 4-terpineol, α β-thujone, 1,8-cineol, verbenol, and verbenone, were tested separately (each diluted 0.5%, vol:vol), 64–72% tick repellency was obtained.

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is an economically important pest of livestock. Previous studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27-kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine whether this protein, now identified as a homolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and whether naïve calves can mount an immune response to it. Calves raised in the winter were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with “natural” Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 d postimmunization in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F(ab′)₂ portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive but that it has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.

Outbreaks of Rift Valley fever (RVF) in Egypt, Yemen, and Saudi Arabia have indicated the potential for this disease to spread from its enzootic areas in sub-Saharan Africa. Because little is known about the potential for most African mosquito species to transmit RVF virus (family Bunyaviridae, genus Phlebovirus, RVFV), we conducted studies to determine the vector competence of selected African species of mosquitoes for this virus. All eight species tested [Aedes palpalis (Newstead), Aedes mcintoshi Huang, Aedes circumluteolus (Theobald), Aedes calceatus Edwards, Aedes aegypti (L.), Culex antennatus (Becker), Culex pipiens (L.), and Culex quinquefasciatus Say], were susceptible to infection, and all except Ae. calceatus, Ae. aegypti and Cx. quinquefasciatus transmitted RVFV by bite after oral exposure. Estimated transmission rates for mosquitoes that successfully transmitted RVFV by bite ranged from 5% for Ae. mcintoshi to 39% for Ae. palpalis for mosquitoes that fed on a hamster with a viremia ≥10⁸ plaque-forming units of virus/ml. We did not recover RVFV from any of 3,138 progeny of infected female mosquitoes. RVFV is unusual among arboviruses in that it has been isolated in nature from a large number of species and that numerous mosquitoes and other arthropods are able to transmit this virus in the laboratory. The recent introduction and spread of West Nile virus into the Americas and the spread of RVFV to the Arabian Peninsula illustrates the potential for viruses, once enzootic in Africa, to spread to other parts of the world.

Mechanical transmission of Anaplasma marginale by horse flies (Tabanidae) is thought to be epidemiologically significant in some areas of the United States. We compared the relative efficiencies of mechanical transmission of Anaplasma marginale by the horse fly, Tabanus fuscicostatus Hine, during acute infection (≈10⁷ to ≈10⁹ infected erythrocytes [IE]/ml blood) with biological transmission by Dermacentor andersoni Stiles in the persistent phase of infection (≈10^2.5 to ≈10⁶ IE/ml). Transmission of A. marginale was not observed when horse flies were partially fed on an acutely infected donor calf and immediately transferred to susceptible calves to complete their blood meal. Ticks that were acquisition fed on the same donor host after it reached the persistent phase of infection successfully transmitted A. marginale when transferred to the same recipient calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >240-fold higher than that from which ticks transmitted with 100% efficiency shows that tick-borne biological transmission is at least two orders of magnitude more efficient than mechanical transmission by horse flies.

Hematophagous Diptera landing on a horse were removed by vacuum, and their numbers were related to a paired carbon dioxide-baited suction trap at three locations in southern California where West Nile virus activity was high during the preceding year. Insects collected from the horse included mosquitoes (nine species), biting midges (Culicoides sonorensis Wirth & Jones), and black flies (Simulium bivittatum Malloch). Mosquitoes were predominantly collected from the head, crest, withers, neck, chest, and shoulders of the horse, whereas biting midges and black flies were predominantly collected from the ventral midline of the horse. Culex erythrothorax Dyar was by far the most abundant mosquito species collected overall. Frequency of engorgement for mosquitoes captured from the horse ranged by species from zero to 58.3%, with Culex pipiens quinquefasciatus Say having the lowest value (16.7% or one of six mosquitoes) of species that fed on the horse. The number of insects captured at the horse and paired CO₂-baited suction trap was not different for Anopheles franciscanus McCracken, Culex tarsalis Coquillett, and S. bivittatum. Cx. p. quinquefasciatus was captured in greater numbers in the CO₂-baited suction trap, whereas Anopheles hermsi Barr & Guptavanji, Cx. erythrothorax, Culiseta inornata (Williston), and Culiseta particeps (Adams) were captured in greater numbers from the horse. The horse biting rate was very low for Cx. p. quinquefasciatus, intermediate for Cx. tarsalis, and very high for Cx. erythrothorax. Both Cx. tarsalis and Cx. erythrothorax should be considered likely epizootic vectors of West Nile virus to horses in rural southern California.

Host-feeding patterns of Culex pipiens L. collected in southwest suburban Chicago in 2005 were studied using polymerase chain reaction (PCR) and DNA sequencing techniques. Culex spp. mosquitoes, most identified to Cx. pipiens and the remainder to Cx. restuans by PCR, had fed on 18 avian species, most commonly American robin (Turdus migratorious), house sparrow (Passer domesticus), and mourning dove (Zenaida macroura). Additional blood meals were derived from four mammal species, primarily humans and raccoons (Procyon lotor). During a West Nile virus (WNV) epidemic in 2005, West Nile virus (WNV) RNA was detected in heads and thoraces of five Cx. pipiens (n = 335, 1.5%) using quantitative PCR. The hosts of these virus-infected, blood-fed mosquitoes included two American robins, one house sparrow, and one human. This is the first report of a WNV-infected Cx. pipiens mosquito collected during an epidemic of WNV that was found to have bitten a human. These results fulfill a criterion for incrimination of Cx. pipiens as a bridge vector.

A TissueLyser system (QIAGEN) was used to rapidly and accurately estimate bluetongue virus “loads” in individual adult Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae). The optimized homogenization program that was developed, involved shaking insects for 1 min at 25 Hz with 2- or 3-mm stainless steel ball bearings. This program was used to measure the quantities of bluetongue virus present in insects that had either been inoculated or had ingested a viremic bloodmeal through an artificial membrane. The virus titers obtained using either infection technique and the optimized program did not differ significantly from those obtained using a polypropylene motor-driven pestle, a method that is currently in common use for studies of vector competence). The advantages of the new method, as a rapid means of detecting fully disseminated infections in individual field-caught flies, are discussed. Its use is compared with the processing of pools of Culicoides by conventional methods, where the extent of dissemination of the virus is unknown and could wrongly implicate species that are of low importance in transmission.

Quantification of Leishmania parasites in the sand fly digestive tract is important for evaluation of vector competence. We compared quantitative polymerase chain reaction (Q-PCR) with two “traditional” methods, estimation in situ and direct counting with the aid of a hemocytometer, to evaluate their usefulness in different parasite–vector combinations. Phlebotomus duboscqi Neveu-Lemarie and Phlebotomus arabicus Theodor sand flies were infected with Leishmania major and Leishmania infantum, respectively, and different approaches were compared to determine the intensity of Leishmania infections before and after defecation of the bloodmeal (on days 2 and 8 postinfection, respectively). Estimation of parasite numbers in situ is only a semiquantitative method, but it is quick and provides data about localization of infection. We recommend this technique for low-intensity infections after the bloodmeal is passed. Counting in a hemocytometer is a suitable technique for heavily infected sand flies or for quantification of Leishmania within the bloodmeal. Because of its relatively high cut-off (60 parasites per gut), it is not useful for low-intensity infection soon after defecation when parasites are attached to midgut. The most accurate approach for parasite quantification in any type of sand fly infection is Q-PCR. This method is also highly sensitive and can detect one parasite per gut. Localization of a Leishmania infection in the sand fly midgut is a parameter equally important to parasite numbers. Therefore, to get full information about the Leishmania development in sand flies, we propose to combine various techniques. Both Q-PCR and counting with a hemocytometer always should be preceded by in situ examination under the microscope to assess the localization of the infection.

Most vector-borne zoonotic pathogens are transmitted among several host species, but different species vary considerably in their importance to pathogen transmission, at least partially because they vary in their propensity to infect feeding vectors. This propensity is often called realized reservoir competence. Realized reservoir competence is the product of 1) the probability the individual host is infected, i.e., infection prevalence, and 2) the probability that if the host is infected, it will transmit the infection to a feeding vector, or infectivity. Prevalence varies in space and time, whereas infectivity may be a property of the host species. Both prevalence and infectivity are ecologically and epidemiologically important, but measuring them simultaneously is difficult. We present a probabilistic model that separately estimates host infection prevalence and infectivity from data on the infection status of vectors collected from individual hosts, data generally used to measure realized reservoir competence. We then consider how imperfect diagnostic tests (i.e., false negatives and positives) influence these probabilities—estimates of prevalence and infectivity are fairly robust to false negatives, but not to false positives. We thus extend the model to estimate the rate of false positives in order to improve estimates of prevalence and infectivity. We illustrate these methods by reanalyzing data from LoGiudice et al. (2003; Proc. Natl. Acad. Sci. U.S.A. 100: 567–571) on the reservoir competence of ten vertebrate hosts of Borrelia burgdorferi, the agent of Lyme disease. We find that these vertebrate hosts vary both in prevalence and infectivity and that both values are highly, positively correlated among species.

Productivity of defective rainwater harvesting structures (RWHS) and other peridomestic habitats for dengue vector was assessed in a coastal town in Tamil Nadu, southern India, where dengue cases were reported. Of 31,709 houses, 792, 790, and 759 were surveyed during southwest monsoon, northeast monsoon, and summer season, and RWHS were found in 651, 638, and 544 houses, respectively. Of these RWHS, 23.3, 34.6 and 14.2% had defects; 20.7, 30.9, and 11.8% were holding water; and 6.5, 11.9, and 5.7% supported dengue vectors. Six types of RWHS, namely, open percolation pit, covered percolation pit, sealed percolation pit, rechargeable trench with bore, and structures connected to well in use or disuse were found. Number of female pupae of Aedes, i.e., pupal productivity obtained from RWHS ranged from 114 in summer to 1,174 in northeast monsoon, and open percolation pit contributed maximum (11.5%). The productivity of habitats other than RWHS varied from 635 in summer to 1,754 in northeast monsoon. Overall, the pupal productivity recorded from RWHS was 30.1% and in other habitats was 69.9%. A hierarchical cluster analysis showed three clusters of 23 types of habitats, which differed significantly in pupal production (F = 426.4, P < 0.05). Cluster I consists of 15 habitats, namely, disused well, RWHS-disused well, RWHS-used well, metal container, coconut shell, glass bottle, sealed percolation pit, used well, ornamental container, tree stump, defrost water collection tray, disposable cup, flower pot, broken toilet ware, and sun shade. Cluster II includes seven habitats, i.e., grinding stone, open percolation pit, covered percolation pit, cement tank, mud pot, plastic container, and rechargeable trench with bore, and cluster III includes automobile tire alone. Cluster II and III contributed to 80.1% of the total pupal production, whereas the remaining 19.9% by cluster I. The study showed that the defective RWHS, particularly open and covered percolation pits were found to be among the key containers, propagating Aedes population. The pupae per person obtained during northeast monsoon in different houses varied between 0.077 and 2.839 (average 0.864). House and Breteau Indices were relatively higher during northeast monsoon, whereas the Container Index was higher in southwest monsoon. In view of risk of dengue vectors propagation, the need for source reduction involving community and prioritizing control measures toward the highly productive water-holdings is discussed.

Infestation of animal tissues by dipteran larvae (myiasis) commonly occurs in many species, but it is unusual for humans in temperate regions. Nevertheless, human myiasis is regularly observed in many primary care facilities in the United States. Beyond medical issues associated with treating human myiasis, both the causal agent and the longevity of myiasis can have legal implications, for example, as evidence of neglect. Cases of human myiasis in the United States typically involve imported myiasis from torsalo, Dermatobia hominis (Linnaeus, Jr.) (Diptera: Oestridae), or facultative myiasis from calliphorids. Here, however, we report two cases of wound myiasis caused by phorid larvae occurred in southeastern Nebraska within 10 mo. Degree-day analysis indicates initial infestation occurred 2 and 3 d before discovery. There are few previous reports of phorid wound myiasis; so, the occurrence of two cases in so short a period suggests that phorids are more important than previously appreciated.

The sand fly Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae), is the vector of the Neotropical parasite Leishmania chagasi. Designing methods to control the spread of this pathogen involves maintaining large laboratory colonies of these flies. However, transmission studies have been hampered particularly in regard to significant fluctuations in colony productivity. In the process of optimizing our sand fly rearing protocol, we increased moisture levels in the environment of the adult blood-fed female and found that egg development increased, whereas sand fly longevity decreased. Interestingly, continuous moisture from the fourth day after a bloodmeal significantly increased the longevity of the fly, but it did not significantly decrease the number of eggs developed. These results will help guide investigators aiming to manipulate the longevity and productivity of sand fly colonies.

Descriptions of morphological anomalies of phlebotomine sand flies (Diptera: Psychodidae) have been recorded in both the New World and the Old World. The majority of these anomalies are associated with the genitalia, the number of cibarial teeth, wing venation, the size of the palps, and other less significant features. Such morphological variations, particularly in symmetry, can result in incorrect species identification. The occurrence of gynandromorphism in sand flies is a rare phenomenon. Currently, only five such cases have been registered in worldwide literature. In Brazil, only one report of gynandromorphism was observed in a specimen of Lutzomyia longipalpis (Lutz & Neiva 1912) from the state of the Ceará. We report here on the first known case of gynandromorphism in Lutzomyia davisi (Root 1934) captured in Alta Floresta, Mato Grosso state, Brazil.

Mosquito collections were done on microfilaremic dogs, positive for Dirofilaria immitis (Leidy), for 15 consecutive nights in Celestun, Yucatan, southeastern Mexico, during January 2007. In total, 275 mosquitoes (3 ♂ and 272 ♀) of five species were collected: Ochlerotatus taeniorhynchus (Wiedemann), Ochlerotatus sollicitans (Walker), Culex quinquefasciatus Say, Culex interrogator (Dyar & Knab), and Aedes aegypti (L.). Oc. taeniorhynchus was the species most frequently collected, and it had the highest rate of feeding success and the highest attack rates. First (L1) and third (L3) instars of Dirofilaria were observed in all mosquito species collected except for Ae. aegypti: 23 of 223 Oc. taeniorhynchus, three of 40 Cx. quinquefasciatus, and one of six Oc. sollicitans and one of one Cx. interrogator were infected with Dirofilaria (10.3% of total examined mosquitoes). This is the first report of Dirofilaria (presumably D. immitis) isolation from wild-caught mosquitoes in Mexico. Results imply that D. immitis can develop from microfilaria to infective L3s in Oc. taeniorhynchus, Cx. quinquefasciatus, and Oc. sollicitans, but L3s (infectious) were only recovered in heads of Oc. taeniorhynchus. Thus, Oc. taeniorhynchus can be considered a potential vector of dirofilariasis in Celestun, because infective L3 Dirofilaria were found in the vicinity of the mouthparts, and this mosquito has a strong association with microfilaremic dogs in this study area.

The microbial flora associated with Anopheles darlingi Root (Diptera: Culicidae), a major Neotropical malaria vector, was investigated for the development of a paratransgenesis-based approach to control malaria transmission in Brazil. Female mosquitoes were collected using human land catches and captured insects provided a bloodmeal. The controlled blood feeding resulted in increased detection of mosquito bacterial population because it was possible to retrieve bacterial DNA from all blood-fed mosquitoes. The 16S sequences of bacteria recovered, include some closely related to those found in other vector mosquitoes, including Aeromonas, Pantoea and Pseudomonas species.

Four hundred and sixty-six questing Amblyomma americanum (L.) (Acari: Ixodidae) from Carolina County, VA, and 98 questing A. americanum from Chatham County, NC, were screened by polymerase chain reaction (PCR) for the Bartonella 16S-23S intergenic spacer region. Two amplicons, ≈270–280 bp, were detected in two ticks from Virginia. Based upon PCR and sequencing, an adult male and adult female tick harbored DNA sequences closely related to Bartonella tamiae (DQ395180). Bartonella DNA was not detected in A. americanum from North Carolina. Potential transmission of Bartonella spp. by A. americanum should be the focus of future experimental studies.