Phlebotomus (Phlebotomus) papatasi (Scopoli) collected in human dwellings from an agricultural villages Chaura, located in Gaya district, Bihar, India, showed morphological and anatomical variations. Male sand flies of this species exhibited variations in the genital structures, while females showed differences in the spermathecae and antenna segment three (A3). When the mitochondrial DNA of both male and female P. (P.) papatasi sensu lato population subjected to DNA barcoding, both the sexes of P. (P.) papatasi variants were found to be associated. The differences in the morphometric characteristics clearly constitutes preliminary evidence for infraspecific variation in P. (P.) papatasi s.l. population in India.

A new species of phlebotomine sand fly found in Minas Gerais state, Brazil, is described and illustrated based on male and female morphological characteristics. The new species belongs to the rupicola series within the subgenus Evandromyia Mangabeira. Both sexes are morphologically close to Evandromyia rupicola (Martins, Godoy & Silva) and Evandromyia grimaldii Andrade Filho, Pinto, Santos & Carvalho and the characteristics for their classification are here presented.

The life history characteristics of the rove beetle Paederus fuscipes Curtis were studied under laboratory conditions using three field strains from Malaysia: Desa Wawasan (DW), Sri Pinang (SP), and Ampang Jajar (AJ). The total development time of immature stages differed significantly among the three strains, especially between DW (17.43 ± 0.16 d), SP (18.60 ± 0.19 d), and AJ (18.68 ± 0.22 d). Adult females and males from DW also exhibited a shorter life span, although the difference among strains was not significant. In terms of fecundity, the numbers of eggs laid per female for DW, SP, and AJ were 121.28 ± 15.98, 127.30 ± 18.01, and 147.45 ± 17.12, respectively. Additionally, because of the shorter life span in DW strain, two apparent peaks in age-stage specific fecundity were detected. The beetles compensated for their shorter life span by increasing their reproductive activity to sustain the progeny in the population. The intrinsic rates of increase (r) of P. fuscipes from DW, SP, and AJ were 0.0773 ± 0.0046 d^-1 0.0788 ± 0.0051 d^-1, and 0.0873 ± 0.0054 d^-1, respectively; and the net reproduction rates (R₀) were 40.09 ± 7.39 offspring, 45.29 ± 8.74 offspring, and 42.34 ± 8.25 offspring, respectively. The mean generation time of P. fuscipes from AJ was 43.08 ± 1.07 d, which was significantly higher than that from DW (47.95 ± 1.36 d) and SP (48.57 ± 1.43 d). The total immature development time of P. fuscipes in this study was shorter than values reported in previous studies.

Reproductive potential was assessed for stable fly cohorts fed cattle, chicken, or horse blood. Flies provided chicken blood oviposited 20% more eggs per day than did those fed cattle or horse blood. However, flies provided cattle or horse blood were fecund 50% longer. When both egg viability and number of eggs produced were considered, lifetime reproductive potential was almost twice as high for flies fed cattle or chicken blood than for flies fed horse blood. Maternal investment, which took egg production and volume into account, was higher in cohorts fed cattle blood (70 mm³) when compared with the other treatments (chicken = 54 mm³, horse = 55 mm³). This is the first report of stable flies producing viable eggs after feeding on bird blood. Results from this study in addition to field observations indicate that stable fly interactions with birds may be limited to relatively low risk scenarios.

Measurement of the survival and dispersal rates of mosquito vectors is an important step in designing and implementing control strategies. Vector survival plays a key role in determining the intensity of pathogen transmission, and vector movement determines the spatial scale on which control efforts must operate to be effective. We provide the first estimates of field survival and dispersal rates for Culex pipiens L. in North America, an important enzootic and bridge vector for West Nile virus (WNV). We conducted mark-release-recapture studies in a residential area near Washington, DC, in two consecutive years and fit nonlinear regression models to the recapture data that incorporate weather information into survival and recapture probabilities. We found that daily survival rates were not significantly different between the 2 yr but were negatively affected by rainfall. The daily survival rate was 0.904 ± 0.037 (SE), which implies an average longevity of 10.4 d. As with other vector-borne pathogens, the measured survival rate suggests that at our site the majority of WNV-infected Cx. pipiens mosquitoes may perish before becoming infectious (being able to transmit WNV to hosts). We found relatively little evidence of dispersal after the initial night after release. Our results suggest that transmission of WNV and other pathogens transmitted by Cx. pipiens may be highly local and they highlight the importance of factors that influence survival of mosquito vectors.

Analysis of molecular genetic diversity in nine marker regions of five genes within the bacteriophage WO genomic region revealed high diversity of the Wolbachia pipentis strain wPip in a population of Culex pipiens L. sampled in metropolitan Chicago, IL. From 166 blood fed females, 50 distinct genetic profiles of wPip were identified. Rarefaction analysis suggested a maximum of 110 profiles out of a possible 512 predicted by combinations of the nine markers. A rank-abundance curve showed that few strains were common and most were rare. Multiple regression showed that markers associated with gene Gp2d, encoding a partial putative capsid protein, were significantly associated with ancestry of individuals either to form molestus or form pipiens, as determined by prior microsatellite allele frequency analysis. None of the other eight markers was associated with ancestry to either form, nor to ancestry to Cx. quinquefasciatus Say. Logistic regression of host choice (mammal vs. avian) as determined by bloodmeal analysis revealed that significantly fewer individuals that had fed on mammals had the Gp9a genetic marker (58.5%) compared with avian-fed individuals (88.1%). These data suggest that certain wPip molecular genetic types are associated with genetic admixturing in the Cx. pipiens complex of metropolitan Chicago, IL, and that the association extends to phenotypic variation related to host preference.

Aquatic containers, including tree holes and vehicle tires, harbor a diverse assemblage of mosquitoes capable of vectoring important diseases. Many studies have examined containers as a mosquito breeding site, although no data exist that have simultaneously compared mosquito communities between tree holes and tires, and few have quantified differences in environmental factors or food resources that may be important for explaining population or community differences. At two times (early and late summer 2009) we sampled two tire and two tree hole sites in south-central Mississippi, and for each container we enumerated mosquito larvae and measured several environmental parameters (canopy cover, water volume, and detritus), and biomass and productivity of fungi and bacteria, and species richness and abundance of protozoans. Tree holes held less water but were more shaded compared with tires; however, after correcting for volume differences, tree holes contained more detritus and were higher in some microorganism measures (protozoan richness, bacterial productivity in the water column). Based on community dissimilarity analysis of mosquitoes, strong differences existed between container types and sampling period; Aedes albopictus (Skuse) and Culex quinquefasciatus (Say) were dominant in tires, whereas Ae. triseriatus (Say) and Orthopodomyia signifera (Coquillett) were dominant in tree holes. This study also reports the use of tires by the invasive mosquito Cx. coronator (Dyar and Knab). Tree holes supported a higher density of larvae but fewer species than tires, though there was variation across time. Our work illustrates that detrital inputs and some microorganisms differ in fundamental ways between tires and tree holes, and because of compositional differences in mosquito communities, these small aquatic habitats cannot be considered to be homogeneous mosquito habitats.

We used high-throughput DNA sequencing to explore bacterial communities of three species of Oropyslla fleas [Oropsylla hirsuta (Baker), Oropsylla montana (Baker), and Oropsylla tuberculata cynomuris (Jellison) ] and detected seven bacterial lineages related to known insect symbionts. No significant co-occurrence patterns were detected among bacterial lineages, but relative abundance data suggest that the two most common lineages (Bartonella and Rickettsiales) interact negatively. Furthermore, presence of these two lineages significantly reduced bacterial diversity within fleas.

Previous results indicate that glycerol-3-phosphate dehydrogenase variability represents the adaptation of Ixodes ricinus L. (Acari: Ixodidae) to fluctuations of environmental conditions, particularly to temperature. Analysis of crucial polymorphisms in I. ricinus Gpdh gene was done by the restriction method, and three different haplotypes were obtained (GPDH₄₄₁ 1, GPDH₄₄₁ 2, and GPDH₄₄₁ 3), corresponding to GPDH alleles detected by allozyme electrophoresis. Differences in GPDH₄₄₁ haplotype and genotype frequencies were found between samples from open and forest habitats. Significant seasonal variations of GPDH₄₄₁ haplotype and genotype frequencies were detected in samples from the open habitats. No seasonal variations were observed at forest localities, probably because of the less pronounced amplitude of environmental factors. The possible role of host availability was discussed as an important factor that affects seasonal dynamics and genetic composition of tick populations.

We investigated the occurrence of spatial structuring in Triatoma infestans (Klug) (Hemiptera: Reduviidae) populations 12 yr after the last community-wide insecticide spraying campaign in rural Pampa del Indio, in the Gran Chaco of northeastern Argentina. In total, 172 male and 149 female right wings collected at 16 georeferenced sites with at least 10 individuals of the same sex were analyzed using geometric morphometry. Mean female body length and wing centroid size (CS) were significantly larger than for males. Log-transformed CS and length were significantly and positively correlated both for males and females. Males collected in domiciles had significantly smaller CS than those collected in peridomestic structures both closed (kitchens or storerooms) or open (chicken coops), in agreement with our previous results elsewhere in the dry Argentine Chaco. Female wing CS was not significantly different between ecotopes. Wing shape analyses showed the occurrence of significant geographic structuring in males and females combined and in males only. Male wings showed a strong association between Mahalanobis distance and geographic distance. In general, Mahalanobis distances were significantly different between collection sites located >4 km apart. For collection sites located <4 km apart, the greater the geographic distance the larger the difference in wing shape variables. Among females, only a partial correspondence between geographic groups and Mahalanobis distances was recorded. The strong spatial structuring found in T. infestans populations may be useful for the identification of putative reinfestation sources after vector control interventions.

Identification of the source of bloodmeals in vectors plays an important role in epidemiological studies by determining the host preferences of wild sand flies in natural habitat. The anthropophilic index is a crucial component in human leishmaniasis. Bloodmeal analysis can identify the reservoir hosts of vector borne diseases. The amplification of the mitochondrial cytochrome b gene, followed by reverse line blot analysis, helps to identify the bloodmeal ingested by the wild caught sand flies. In the current study, blood fed sand flies were collected from three different villages in Bihar, India, by using Centers for Disease Control mini traps with incandescent light. Traps were placed in five different sites in the villages. Whole genome DNA was extracted from the blood fed sand flies and was amplified for the cytochrome b region, followed by reverse line blot analysis. In total, 442 blood fed sand flies were analyzed out of which 288 (65%) were positive to cytochrome b polymerase chain reaction. Humans, cattle, buffalo, and goats were the major bloodmeals identified, followed by chickens. In some of the blood fed sand flies, multiple bloodmeals were identified. In the current study, sand flies mostly fed on humans, followed by cattle, buffalo, and goats. In this regard, it is necessary to also consider cattle, buffalo, and goats when addressing vector control in Bihar, India.

Culex erraticus (Dyar & Knab) is a potential competent vector for several arboviruses such as Eastern and Venezuelan equine encephalitis viruses and West Nile virus. It therefore may play a role in the maintenance and spread of viral populations in areas of concern, including the United States where it occurs in >33 states. However, little information is available on potential barriers to movement across the species' distribution. Here, we analyze genetic variation among Cx. erraticus collected from Colombia, Guatemala, and nine locations in the United States to better understand population structure and connectivity. Comparative sequence analysis of the second internal transcribed spacer and mitochondrial NADH dehydrogenase genes identified two major lineages of sampled populations. One lineage represented the central and eastern United States, whereas the other corresponded to Central America, South America, and the western United States. Hierarchical analysis of genetic variation provided further evidence of regional population structure, although the majority of genetic variation was found to reside within populations, suggestive of large population sizes. Although significant physical barriers such as the Chihuahuan Desert probably constrain the spread of Cx. erraticus, large population sizes and connectivity within regions remain important risk factors that probably contribute to the movement of arboviruses within and between these regions.

The bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), has experienced an extraordinary global resurgence in recent years, the reasons for which remain poorly understood. Once considered a pest of lower socioeconomic classes, bed bugs are now found extensively across all residential settings, with widespread infestations established in multiapartment buildings. Within such buildings, understanding the population genetic structure and patterns of dispersal may prove critical to the development of effective control strategies. Here, we describe the development of 24 high-resolution microsatellite markers through next generation 454 pyrosequencing and their application to elucidate infestation dynamics within three multistory apartment buildings in the United States. Results reveal contrasting characteristics potentially representative of geographic or locale differences. In Raleigh, NC, an infestation within an apartment building seemed to have started from a single introduction followed by extensive spread. In Jersey City, NJ, two or more introductions followed by spread are evident in two buildings. Populations within single apartments in all buildings were characterized by high levels of relatedness and low levels of diversity, indicative of foundation from small, genetically depauperate propagules. Regardless of the number of unique introductions, genetic data indicate that spread within buildings is extensive, supporting both active and human-mediated dispersal within and between adjacent rooms or apartments spanning multiple floors.

Ticks are efficient ectoparasites that are able to steal blood, a rich source of nutrients, from their vertebrate hosts. The nymphal developmental stage of ticks plays an important role for pathogen transmission to human and other animal hosts. In this article, we describe a bloodmeal-based sex differentiation tool to generate adult female ticks infected with Ehrlichia chaffeensis to investigate vector-pathogen interactions (functional genomics and gene expression studies). We demonstrate that there is a correlation between the uptake of blood during nymph attachment and the molting into male or female adult ticks. The data obtained from the bloodmeal experiments suggest that nymphs that molt into females presumably imbibe more blood than those that become male during the nymphal stage. The natural low E. chaffeensis infection rate in female adult Amblyomma americanum (L.) is a major limiting factor to investigate Ehrlichia-Amblyomma interactions. To generate Ehrlichia-infected female adult ticks, we inoculated obligate E. chaffeensis (Arkansas strain) infected DH82 cells into heavier engorged nymphs (>12 mg) and allowed them to molt. Freshly molted adults were used to test the E. chaffeensis infection rate. E. chaffeensis genomic DNA was extracted from individual unfed and partially blood fed tick midgut and salivary gland tissues. The tissue samples were tested for the presence of E. chaffeensis using the nested polymerase chain reaction process. Polymerase chain reaction-amplified fragments were detected in unfed and partially fed tissues, demonstrating successful E. chaffeensis infection of tick tissues. This method was used to successfully show differential expression of selected tick genes in E. chaffeensis-infected midguts and salivary glands.

Acetylcholinesterase cDNAs, BmAChE1, BmAChE2, and BmAChE3 of Rhipicephalus (Boophilus) microplus (Canestrini) were sequenced and found to exhibit significant polymorphism. A portion of the predicted amino acid substitutions in BmAChE1, BmAChE2, and BmAChE3 were found predominantly in organophosphate-resistant strains, but most did not correlate with resistant status. Multiple transcripts were observed from individual ticks, suggesting possible gene duplication or alternative splicing to produce more than two transcripts per individual. BmAChE1 transcript polymorphisms associating with organophosphate-resistant status in laboratory strains were surveyed in laboratory and Mexican strains of R. microplus by sequencing BmAChE1 genomic DNA. Quantitative real-time polymerase chain reaction was used to determine copy numbers of BmAChE1 (eight copies/ haploid genome), BmAChE2 (16 copies/ haploid genome), and BmAChE3 (four copies/ haploid genome). Presence of at least three highly polymorphic amplified genes expressing AChE in tick synganglion suggested that ticks maintain a large and diverse assortment of AChE alleles available for rapid recombination and selection, which potentially reduces fitness costs associated with individual mutations. Elevated copy numbers for each of the BmAChEs may also explain previous failures to identify mutations resulting in insensitivity to organophosphates. It is clear that development of phenotypic resistance to organophosphates is highly complex and may be multigenic in character.

The kissing bug Triatoma rubida (Uhler, 1894) is found in southwestern United States and parts of Mexico where it is found infected with Trypanosoma cruzi, invades human dwellings and causes allergies from their bites. Although the protein salivary composition of several triatomine species is known, not a single salivary protein sequence is known from T. rubida. Furthermore, the salivary diversity of related hematophagous arthropods is very large probably because of the immune pressure from their hosts. Here we report the sialotranscriptome analysis of T. rubida based on the assembly of 1,820 high-quality expressed sequence tags, 51% of which code for putative secreted peptides, including lipocalins, members of the antigen five family, apyrase, hemolysin, and trialysin families. Interestingly, T. rubida lipocalins are at best 40% identical in primary sequence to those of T. protracta, a kissing bug that overlaps its range with T. rubida, indicating the diversity of the salivary lipocalins among species of the same hematophagous genus. We additionally found several expressed sequence tags coding for proteins of clear Trypanosoma spp. origin. This work contributes to the future development of markers of human and pet exposure to T. rubida and to the possible development of desensitization therapies. Supp. Data 1 and 2 (online only) of the transcriptome and deducted protein sequences can be obtained from  http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S1-web.xlsx and  http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S2-web.xlsx.

Odorant binding proteins (OBPs) play a critical role in mediating mosquito behaviors. In the current study, four AealOBP genes were cloned and sequenced. Basic Local Alignment Search Tool Program and phylogenetic analysis of the deduced amino acid sequences identified a unique putative ortholog in Aedes aegypti (L.) for each Aedes albopictus (Skuse) OBP. Quantitative analysis showed some AealOBPs with a strong female/ male expression ratio, which we proposed to be involved in the host seeking of female mosquitoes. Other OBPs are expressed at higher levels in male antennae and are considered candidates for the detection of floral volatiles. The current study provides basis for further detailed molecular characterization of Ae. albopictus olfactory systems.

Culex theileri Theobald (Diptera: Culicidae) is one of the most common mosquito species in northeastern Turkey and serves as a vector for various zoonotic diseases including West Nile virus. Although there have been some studies on the ecology of Cx. theileri, very little genetic data has been made available. We successfully sequenced 11 gene fragments from Cx. theileri specimens collected from the northeastern part of Turkey. On average, we found a Single nucleotide polymorphism every 45 bp. Transitions outnumbered transversions, at a ratio of 2:1. This is the first report of genetic polymorphisms in Cx. theileri and Single nucleotide polymorphism discovered from this study can be used to investigate population structure and gene-environmental interactions.

This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L.) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirmed to be an insect AChE2-type enzyme with substrate preference for acetylthiocholine (K_m 31.3 µM) over butyrylthiocholine (K_m 63.4 µM) and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine (2.3 × 10 ^-10 M), BW284c51 (3.4 × 10^-8 M), malaoxon (3.6 × 10^-8 M), and paraoxon (1.8 × 10^-7 M), and was less sensitive to the butyrylcholinesterase inhibitors ethopropazine (1.1 × 10 ^-6M) and iso-OMPA (4.1 × 10 ^-4 M). rHiAChE containing the G262A substitution exhibited decreased substrate affinity for both acetylthiocholine (K_m 40.9 µM) and butyrylthiocholine (K_m 96.3 µM), and exhibited eight-fold decreased sensitivity to paraoxon, and ∼1.5- to 3-fold decreased sensitivity to other inhibitors. The biochemical kinetics are consistent with previously reported bioassay analysis, suggesting that the G262A mutation contributes to, but is not solely responsible for observed phenotypic resistance to diazinon or other organophosphates.

The requirement for efficient mosquito mass rearing technology has been one of the major obstacles preventing the large scale application of the Sterile Insect Technique against mosquitoes. At the Food and Agriculture Organization/International Atomic Energy Agency (FAO/IAEA) Insect Pest Control Laboratories we developed a larval rearing unit based on the use of a stainless steel rack that operates 50 thermoformed ABS plastic trays and is expected to be able to successfully rear 140,000–175,000 Anopheles arabiensis (Patton) adult mosquitoes per rack. The mechanized rearing unit is simple to handle, maintains minimal water temperature variation and negligible water evaporation and allows normal larval development. The mosquito mass-rearing tray was designed to provide a large surface area of shallow water that would closely mimic natural breeding sites. The trays stack into a dedicated rack structure and filling and draining were easily performed. The close stacking of the trays in the rack and the possibility to tightly line up several racks makes this rearing unit a valid solution for maximal use of the space thus reducing construction, heating, and cooling costs. The low amount of labor required to operate the system also reduces labor costs that represent one of the main expenditures in any mass rearing facility operation. Preliminary experiments performed on Aedes albopictus (Skuse) also confirm the possibility of successfully extending the use of this technology to other mosquito species. Our larval rearing unit could enhance any mosquito control strategy in which large-scale releases of mosquitoes are needed to suppress or replace natural populations.

Pyriproxyfen is an insect growth regulator with juvenile hormone-like activity that has potential uses for dipterans that are difficult to manage with conventional insecticides, such as house flies (Musca domestica L.). The objectives of this study were to determine the efficacy of this insect growth regulator against house flies using variety of delivery systems and target life stages, including an evaluation of the potential for autodissemination by female flies to larval development sites. Adult female house flies exposed to filter paper (3.75% active ingredient) or sugar treated with pyriproxyfen (0.01–0.1%) produced significantly fewer F₁ pupae than untreated flies. Adult emergence from pupae was unaffected. In contrast, treatment of larval rearing medium with 0.35 ml/cm² of a 12 mg pyriproxyfen /liter preparation had no effect on the number of pupae developing from eggs but markedly inhibited adult emergence from those pupae. There was little difference in susceptibility between an insecticide-susceptible and a wild strain of house fly. The LC₅₀ for inhibiting fly emergence of dust formulations in diatomaceous earth incorporating commercial pyriproxyfen products ranged from 8 to 26 mg/liter, with little difference among products. Compared with untreated flies, significantly fewer pupae were produced at concentrations >0.5% and no adults were produced at concentrations >0.05% pyriproxyfen. When gravid females were exposed for 1 h to treated fabric (6 mg pyriproxyfen/cm²) and allowed to oviposit in rearing media containing eggs, sufficient pyriproxyfen was autodisseminated to reduce adult emergence from those eggs by >99%. Intermittent contact with treated fabric over 2 d reduced adult emergence by 63–76%.

The biochemical mechanisms underlying the increased toxicity of several plant essential oils (thymol, eugenol, pulegone, terpineol, and citronellal) against fourth instar of Aedes aegypti L. when exposed simultaneously with piperonyl butoxide (PBO) were examined. Whole body biotransformational enzyme activities including cytochrome P450-mediated oxidation (ethoxyresorufin O-dethylase [EROD]), glutathione S-transferase (GST), and β-esterase activity were measured in control, essential oil-exposed only (single chemical), and essential oil PBO (10 mg/liter) exposed larvae. At high concentrations, thymol, eugenol, pulegone, and citronellal alone reduced EROD activity by 5–25% 16 h postexposure. Terpineol at 10 mg/liter increased EROD activity by 5 ± 1.8% over controls. The essential oils alone reduced GST activity by 3–20% but PBO exposure alone did not significantly affect the activity of any of the measured enzymes. All essential oils in combination with PBO reduced EROD activity by 58–76% and reduced GST activity by 3–85% at 16 h postexposure. This study indicates a synergistic interaction between essential oils and PBO in inhibiting the cytochrome P450 and GST detoxification enzymes in Ae. aegypti.

The wMelPop strain of Wolbachia is currently being investigated for its potential use as a biological control agent to reduce the ability of Aedes aegypti (L.) mosquitoes to transmit dengue viruses. The survival of a potential wMelPop infected Ae. aegypti strain for field release is important as a higher susceptibility to predation in the wMelPop strain could result in difficulties in achieving fixation. We investigated immature and adult survival as a function of susceptibility to predation by six naturally occurring predator species; cyclopoid copepods, fish, predatory Toxorhynchites mosquito larvae and a salticid jumping spider. The trials indicated that wMelPop infected and uninfected Ae. aegypti larvae and adults were equally susceptible to predation to all six tested predators. In addition to evaluating any potential fitness costs to the infected host, we were unable to demonstrate horizontal transfer of wMelPop via consumption of infected Ae. aegypti larvae to the above predators. That susceptibility to predation was consistent across mosquito life stage, predator species and experimental venue is strong evidence that despite the neurotrophic and extensive nature of wMelPop infection, behavioral changes are not occurring, or at least not a determining factor in survival when exposed to a predator. Based on our results and the ecology of Wolbachia and mosquito predators, horizontal transfer of wMelPop from Ae. aegypti into naturally occurring predators is not cause for concern.

We assessed the toxicity of 17 steam distillate constituents of Cyperus rotundus (L.) rhizome, another seven known compounds of C. rotundus rhizome, and 14 structurally related compounds to females from an insecticide-susceptible KSS strain and two field-collected SEL and DJN colonies of Blattella germanica (L.). High contact fumigant toxicity to KSS females was produced by p-cymene, nerol, linalool, o-cymene, (S)-(-)-citronellal, (1S)-(-)-camphor, terpinolene, and m-cymene (LD₅₀, 0.29–0.47 mg/cm²). The toxicity of these compounds was virtually identical against females from any of the three strains, even though SEL and DJN females were resistant to six acetylcholinesterase inhibitors and three pyrethroids (resistance ratio, 9–154 and 12–195). These results indicate that the compounds and insecticides do not share a common mode of action or elicit cross-resistance. The test compounds were effective in closed but not in open containers against SEL females, indicating that their route of insecticidal action was largely a result of vapor action. Structure-activity relationship indicates that structural characteristics, such as types of functional groups, appear to play a role in determining the terpenoid toxicities to B. germanica. C. rotundus rhizome steam distillate constituents and related compounds described merit further study as potential fumigants for the control of resistant cockroach populations in light of global efforts to reduce the level of highly toxic synthetic insecticides in indoor environments.

The quantity of mosquito larval habitat in a specified area was assessed by placing a known number of ovitraps in the same area. The ovitraps competed for oviposition with the unknown quantity of larval habitat, and that quantity was deduced by comparing the number of eggs laid in the ovitraps when the number of ovitraps was changed from one time to another. This method can be used to assess the effectiveness of source reduction. It also provides key information for using ovitraps to eradicate local mosquito populations by competing with existing breeding sites for oviposition. The same ovitraps that are used for control can provide information on how many ovitraps are necessary to ensure eradication.

A granular formulation of novaluron (Novaluron 0.2G, 0.2% [AI]), a newer benzoylphenyl urea insecticide, was evaluated for its efficacy in controlling the larval stage of horn flies, Haematobia irritans (L.); house flies, Musca domestica L.; and stable flies, Stomoxys calcitrans (L.), in cow manure. Various rates and insecticide placement locations (top, middle, and bottom of manure) were evaluated in this study and all combinations of these variables reduced adult emergence of all three species when compared with the untreated controls. The presence of deformed pupae indicated that novaluron had an insect growth regulator effect on the developing fly larvae. Top, middle, or bottom application rates of 0.125, 0.195, 0.25, and 0.375 g novaluron onto manure samples, reduced adult horn fly emergence by >90%. Middle and bottom application rates of 0.195, 0.25, and 0.375 g novaluron reduced adult house fly emergence >93%. All rates and placement combinations resulted in >98% reduction of adult stable fly emergence. The level of control efficacy observed against these three fly species along with the ease of use of a granular formulation, make this product an ideal candidate for use in an integrated livestock pest management program.

Repellent efficacy of the Off! Clip-on Mosquito Repellent device (S. C. Johnson and Son, Inc., Racine, WI) containing Metofluthrin was evaluated on six human volunteers against the container-breeding mosquito Aedes albopictus (Skuse) and the salt marsh mosquito Aedes taeniorhynchus (Wiedemann) at two field locations in northeastern Florida. The device repelled mosquitoes by releasing a vaporized form of the pyrethroid insecticide metofluthrin ([AI] 31.2%) and provided 70% protection from Ae. albopictus bites for >3 h. For the second field trial, a repellent device that was used in the first trial was tested after being open for >1 wk. This device provided 79% protection from Ae. taeniorhynchus bites for 3 h. Our field results showed that the repellent device was 70 and 79% effective at repelling Ae. albopictus and Ae. taeniorhynchus from human test subjects in both field locations in northeastern Florida.

Peptidoglycan recognition proteins (PGRPs) are a group of proteins that are responsible for the recognition and, in some cases, binding of peptidoglycan (PGN), a unique cell wall component of bacteria, and initiation of immune responses to various types of pathogens. In the current study, full-length cDNA sequences of multiple PGRPs, identified via a database search, were cloned in the mosquito Armigeres subalbatus (Coquillett). During cloning, a novel transcript variant (isoform) of AsPGRP-LC (As: Ar. subalbatus) was also identified that shares a large 5′ end fragment with AsPGRP-LC All four AsPGRP genes (six transcripts) contain a conserved PGRP domain, an ortholog of the amidase-2 domain. Based on predicted functional domain, the six Ar. subalbatus PGRPs resemble both short (AsPGRP-S1) and long (AsPGRP-LBa, AsPGRP-LBb, AsPGRP-LCa, AsPGRPLCb, and AsPGRP-LE) forms of PGRPs as in other insects. Sequence alignments showed that PGRPs are conserved across Dipterans. Phylogenetic analysis indicated that PGRPs represent an ancient gene family that has primarily diverged through speciation events among these Dipterans, with only a limited number of lineage specific gene duplications. Developmental profiling of the six AsPGRP transcripts using real-time polymerase chain reaction revealed that AsPGRP-LCa and AsPGRP-LCb are constitutively expressed at high levels in all developmental stages, while AsPGRP-S1, AsPGRP-LBa, AsPGRP-LBb, and AsPGRP-LE transcripts have low expression in most of the life stages and are increased only at certain times. Tissue profiling of the six AsPGRP transcripts showed that they are expressed in various patterns, even between the different isoforms of the same PGRP gene, indicating that these AsPGRPs may play different functions.

The objective of this study was to investigate the effects of three fragrance fixatives, Glucam P-20, Vanillin, and Fixolide, on the mosquito repellent property of citronella oil lotions. In the current study, two formulae (A and B) of oil-in-water citronella oil lotions were formulated using different ingredients (emulsifiers [Cremophors or Emulwax], stiffening agents, and emollients). Citronella oil was used at 10% wt:wt. The weight ratios tested between citronella oil and each fixative were 1:0.25, 1:0.5, and 1:1. Overall, 20 formulations, including one with no fixatives for both A and B, were produced, A1–A10 and B1–B10. The repellent activities of these 20 lotions against Aedes aegypti (L.) were tested using a human-bait technique. The types and concentrations of fixatives as well as the compositions of the formulations did affect the protection time of the citronella oil lotions. The lotion containing Emulwax and 5% vanillin (B6) was the most effective repellent. It provided the longest protection time of 4.8 h, which exceeded the minimum requirement of 2 h set by the National Institute of Health, Thailand. The shortest protection time (1 h) was observed in the lotion containing Emulwax and 2.5% Glucam P-20 (B2). It could be concluded that the tested fixatives affected the repellent activity of the citronella oil lotions.

Vector competence studies for West Nile virus (WNV) were conducted for two Culex (Culex) restuans Theobald populations Edison Park (EP) and Illinois Medical District (IMD), in Chicago, IL. The aim was to determine if there were differences between mosquito populations that contributed to the observed differences in the prevalence of WNV. Percentages of orally infected, disseminated, and transmitting mosquitoes were estimated using a generalized linear mixed effects model including a random effect for family to account for anticipated within-family correlation. Analysis indicated that percentages of infected, disseminated, and transmitting mosquitoes were not significantly different between EP and IMD. The within-family correlation was 0.46 (95% CI 0.28, 0.67), indicating reasonably strong tendency for WNV titers of bodies, saliva, and legs within families to be similar. Overall, our results show that vector competence of Cx. restuans for WNV is not a contributing factor to the observed differences in WNV human cases between the EP and IMD areas of Chicago.

The host preference of a vector mosquito species plays a significant role in determining human and animal risk of infection with mosquito-transmitted pathogens. Host preferences of common southern California Culex species for four bird species, American crow (Corvus brachyrhynchos), house sparrow (Passer domesticus), house finch (Carpodacus mexicanus), and mourning dove (Zenaida macroura), were examined by determining the proportion of each mosquito species that successfully engorged on each of the four bird species presented equally within a net trap to wild host-seeking mosquitoes. Bloodmeals in engorged mosquitoes captured within the net trap were identified to avian species by using a multiplex polymerase chain reaction assay targeting the cytochrome b gene sequence. There were significant differences in host selection by all three Culex species captured in numbers sufficient for analysis, with Culex erythrothorax Dyar preferentially biting American crows, Culex tarsalis Coquillett preferentially biting house sparrows, and Culex quinquefasciatus Say preferentially biting house finches. All three Culex species demonstrated more frequent engorgement on passerine birds (sparrows, finches, and crows) than the nonpasserine mourning dove. A greater preference for passerine birds might be expected to increase the transmission of pathogens, such as West Nile virus, to which passerine birds are particularly competent hosts.

Determining the host preference of vector ticks is vital to elucidating the eco-epidemiology of the diseases they spread. Detachment of ticks from captured hosts can provide evidence of feeding on those host species, but only for those species that are feasible to capture. Recently developed, highly sensitive molecular assays show great promise in allowing host selection to be determined from minute traces of host DNA that persist in recently molted ticks. Using methods developed in Europe as a starting-point, we designed 12S rDNA mitochondrial gene probes suitable for use in a reverse line blot (RLB) assay of ticks feeding on common host species in the eastern United States. This is the first study to use the 12S mitochondrial gene in a RLB bloodmeal assay in North America. The assay combines conventional PCR with a biotin-labeled primer and reverse line blots that can be stripped and rehybridized up to 20 times, making the method less expensive and more straightforward to interpret than previous methods of tick bloodmeal identification. Probes were designed that target the species, genus, genus group, family, order, or class of eight reptile, 13 birds, and 32 mammal hosts. After optimization, the RLB assay correctly identified the current host species for 99% of ticks [Amblyomma americanum (L.) and eight other ixodid tick species] collected directly from known hosts. The method identified previous-host DNA for approximately half of all questing ticks assayed. Multiple bloodmeal determinations were obtained in some instances from feeding and questing ticks; this pattern is consistent with previous RLB studies but requires further investigation. Development of this probe library, suitable for eastern U.S. ecosystems, opens new avenues for eco-epidemiological investigations of this region's tick—host systems.

The aim of the study was to evaluate rickettsial infection in ticks from wild birds of the Semidecidual and Atlantic Rainforest remnants of three municipalities of the State of Paraná, southern Brazil. Overall, 53 larvae and nymphs collected from birds were checked for the presence of Rickettsia DNA by molecular tests. Five tick species were tested: Amblyomma aureolatum (Pallas), Amblyomma calcaratum Neumann, Amblyomma longirostre (Koch), Amblyomma ovale Koch, and Amblyomma parkeri Fonseca and Aragão. A. longirostre ticks were infected with the spotted fever group agents Rickettsia amblyommii strain AL (32.3% infection rate) and Rickettsia parkeri strain NOD (5.9% infection rate). A new rickettsial genotype was detected in the tick A. parkeri (50% infection rate), which had never been reported to be infected by rickettsiae. Through phylogenetic analysis, this new genotype, here designated as strain ApPR, grouped in a cluster composed by different strains of Rickettsia africae, Rickettsia sibirica, and R. parkeri. We consider strain ApPR to be a new genotype of R. parkeri. This study reports for the first time rickettsial infection in ticks from birds in southern Brazil. The role of migrating birds in the dispersal of these rickettsial strains should be considered in ecological studies of spotted fever group agents in Brazil.

The host blood-feeding patterns of mosquito vectors affects the likelihood of human exposure to zoonotic pathogens, including West Nile Virus (family Flaviviridae, genus Flavivirus, WNV). In Portugal, data are unavailable regarding the blood-feeding habits of common mosquito species, including Culex pipiens L., considered the primary vector of WNV to humans. The sources of bloodmeals in 203 blood-fed mosquitoes of nine species collected from June 2007 to November 2010 in 34 Portuguese counties were analyzed by sequencing cytochrome-b partial fragments. Cx. pipiens was the most common species collected and successfully analyzed (n = 135/ 78). In addition, blood-fed females of the following species were analyzed: Ochlerotatus caspius Pallas (n = 20), Culex theileri Theobald (n = 16), Anopheles maculipennis s.l. Meigen (n = 10), Culiseta longiareolata Macquart (n = 7), Aedes aegypti L. (n = 6), Culex perexiguus Theobald (n = 3), Culiseta annulata Schrank (n = 3), and Ochlerotatus detritus Haliday (n = 3). The Cx. pipiens mosquitoes fed predominantly on birds (n = 55/78, 70.5%), with a high diversity of avian species used as hosts, although human blood was identified in 18 specimens (18/78, 23.1%). No significant differences were found between the host-feeding patterns of blood-fed Cx. pipiens collected in residential and nonresidential habitats. The occurrence of human derived blood meals and the presence of a mix avian—human bloodmeal accordingly suggest this species as a potential vector of WNV. Therefore, in Portugal, Cx. pipiens may play a role both in the avian-to-avian enzootic WNV cycle and in the avian-to-mammal transmission. In this context, the identity of Cx. pipiens (considering the forms molestus and pipiens) and the potential consequence on feeding behavior and WNV transmission are discussed.

Understanding the processes that affect Aedes aegypti (L.) (Diptera: Culicidae) may serve as a starting point to create and /or improve vector control strategies. For this purpose, we performed statistical modeling of three entomological surveys conducted in Clorinda City, northern Argentina. Previous ‘basic’ models of presence or absence of larvae and/or pupae (infestation) and the number of pupae in infested containers (productivity), mainly based on physical characteristics of containers, were expanded to include variables selected a priori reflecting water use practices, vector-related context factors, the history of chemical control, and climate. Model selection was performed using Akaike's Information Criterion. In total, 5,431 water-holding containers were inspected and 12,369 Ae. aegypti pupae collected from 963 positive containers. Large tanks were the most productive container type. Variables reflecting every putative process considered, except for history of chemical control, were selected in the best models obtained for infestation and productivity. The associations found were very strong, particularly in the case of infestation. Water use practices and vector-related context factors were the most important ones, as evidenced by their impact on Akaike's Information Criterion scores of the infestation model. Risk maps based on empirical data and model predictions showed a heterogeneous distribution of entomological risk. An integrated vector control strategy is recommended, aiming at community participation for healthier water use practices and targeting large tanks for key elements such as lid status, water addition frequency and water use.

Human and equine outbreaks caused by eastern equine encephalomyelitis virus (EEEV) typically occur in North America adjacent to coastal wetlands associated with the presence of Culiseta melanura (Coquillet) mosquitoes. Eastern equine encephalomyelitis (EEE) is an emerging disease in Tennessee, as the first records of equine disease began in 2002. In 2006 we trapped and tested mosquitoes for EEEV at hardwood swamps in western Tennessee that were at the epicenter of a multi-equine outbreak in fall of 2005. Additionally, the Tennessee Valley Authority tested mosquito pools collected in Tennessee swamps from 2000 to 2007 for the presence of arboviruses. Two pools of EEEV positive Culex erraticus (Dyer and Knab) mosquitoes were found (one each in 2003 and 2004) in a county adjacent to where the 2005 outbreak occurred. In 2008, another EEE outbreak involving multiple horses occurred in West Tennessee. A brain specimen was collected from a horse during this outbreak and the first isolate of EEEV from Tennessee was obtained. In total, 74,531 mosquitoes collected from 2000 to 2008 were tested via polymerase chain reaction and VecTest for EEEV. The traditional enzootic vector, Cs. melanura, was found in low numbers at all collection sites. Cx. erraticus, however, was consistently found in high numbers and was the only mosquito species in which EEEV was detected. We suggest that EEE transmission may be maintained by Cx. erraticus in a nontraditional cycle. We discuss the importance of a nontraditional cycle from the perspective of EEEV adaptation and emergence.

The increased cases of cutaneous leishmaniasis vectored by Phlebotomus papatasi (Scopoli) in Libya have driven considerable effort to develop a predictive model for the potential geographical distribution of this disease. We collected adult P. papatasi from 17 sites in Musrata and Yefern regions of Libya using four different attraction traps. Our trap results and literature records describing the distribution of P. papatasi were incorporated into a MaxEnt algorithm prediction model that used 22 environmental variables. The model showed a high performance (AUC = 0.992 and 0.990 for training and test data, respectively). High suitability for P. papatasi was predicted to be largely confined to the coast at altitudes <600 m. Regions south of 30° N latitude were calculated as unsuitable for this species. Jackknife analysis identified precipitation as having the most significant predictive power, while temperature and elevation variables were less influential. The National Leishmaniasis Control Program in Libya may find this information useful in their efforts to control zoonotic cutaneous leishmaniasis. Existing records are strongly biased toward a few geographical regions, and therefore, further sand fly collections are warranted that should include documentation of such factors as soil texture and humidity, land cover, and normalized difference vegetation index (NDVI) data to increase the model's predictive power.

Eastern Equine Encephalitis virus (EEEV; family Togaviridae, genus Alphavirus) a highly pathogenic mosquito-borne virus is endemic to eastern North America. The ecology of EEEV in Florida differs from that in other parts of the United States; EEEV in the northeastern United States is historically associated with freshwater wetlands. No formal test of habitat associations of EEEV in Florida has been reported. Geographical Information Sciences (GIS) was used in conjunction with sentinel chicken EEEV seroconversion rate data as a means to examine landscape features associated with EEEV transmission in Walton County, FL. Sentinel sites were categorized as enzootic, periodically enzootic, and negative based on the number of chicken seroconversions to EEEV from 2005 to 2009. EEEV transmission was then categorized by land cover usage using Arc GIS 9.3. The land classification data were analyzed using the Kruskal—Wallis test for each land use class to determine which habitats may be associated with virus transmission as measured by sentinel chicken seroconversion rates. The habitat class found to be most significantly associated with EEEV transmission was tree plantations. The ecological factor most commonly associated with reduced levels of EEEV transmission was vegetated nonforest wetlands. Culiseta melanura (Coquillett), the species generally considered to be the major enzootic EEEV vector, was relatively evenly distributed across all habitat classes, while Aedes vexans (Meigen) and Anopheles crucians Weidemann were most commonly associated with tree plantation habitats.

Truck trap collections of Culicoides biting midges (Diptera: Ceratopogonidae) were made during 2 yr of sampling from 2008 to 2009 at a farm site in southern England. Samples were collected from 810 sample runs carried out over 52 d and contained 7,095 Culicoides of which more than half (50.3%) were identified as Culicoides obsoletus Meigen by using a multiplex polymerase chain reaction assay. Other commonly encountered species included Culicoides scoticus Downes & Kettle (14.7% of total Culicoides caught), Culicoides dewulfi Goetghebuer (3.7%), and Culicoides chiopterus Meigen (4.2%). The activity rates of these species were examined with regard to both meteorological factors (light intensity, humidity, temperature, and wind speed and direction) and other potentially contributing variables (lunar phase and brightness, sunset time, and year) by using generalized linear models. All the species examined were collected in greater abundance at sunset, although the relationship between underlying light intensity and numbers was less pronounced in C. dewulfi and C. chiopterus. Collections of Culicoides were reduced at temperatures above 21°C and were inversely related to wind speed. Variation between species was recorded, however, in response to wind direction: C. dewulfi and C. chiopterus were associated with prevailing winds passing through fields containing livestock, whereas C. obsoletus and C. scoticus demonstrated no such relationship. A male:female ratio of 1:3.56 was observed in catches, and male populations were protandrous. These results are discussed with reference both to the ecology of these species and methods currently used to predict adult Culicoides movement and abundance in Europe.

Rickettsia species are the causative agent of different forms of spotted fever and thus, monitored in a number of prevalence studies. The current study examined the status of ticks from the city of Hanover, Northern Germany, regarding the presence of Rickettsia spp. and coinfections with Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum. In total, 1,089 questing Ixodes ricinus L. ticks were analyzed using quantitative real time polymerase chain reaction. A duplex quantitative real time polymerase chain reaction for simultaneous detection of Rickettsia spp. and Ixodes spp.-DNA as positive control for successful DNA-isolation was established. Rickettsia spp. were detected in 363 (33.3%) of the 1,089 investigated ticks. Quantification of Rickettsia showed that larvae contained up to 50,000 bacteria, nymphs up to 85 million and adults up to 200 million per tick. Species differentiation was possible in 178 out of 363 Rickettsia positive samples and resulted in a predominant occurrence of R. helvetica (98.9%, 176/178), whereas R. monacensis was rarely found (1.1%, 2/178). Besides detection of Rickettsia, positive ticks were compared with results from previous studies to examine coinfections with B. burgdorferi sl and A. phagocytophilum. The resulting coinfection rates were 9.1% (99/1,089) for B. burgdorferi sl and 2.8% (11/391) for A. phagocytophilum. Triple-infection with Rickettsia spp., B. burgdorferi sl, and A. phagocytophilum occurred in 5 (1.3%) out of 391 ticks. The current study is the first presenting quantitative data concerning the load of Ixodes ticks with Rickettsia individuals.

Unique oligonucleotide probes were synthesized to distinguish among closely related vertebrate mitochondrial rDNA sequences present in residual bloodmeals in emergent Amblyomma americanum (L.) (Acari: Ixodidae) nymph life-stage ticks. Use of these probes enabled the identification of the Eastern gray squirrel as an important bloodmeal source in nymphs harboring Ehrlichia and Borrelia species. These results were confirmed by identifying these same bacterial genera in Eastern gray squirrel tissues.

The genes period (per) and timeless (tim) are core components of the circadian clock that regulates a wide range of rhythmic biochemical, physiological, and behavioral processes in prokaryotes and eukaryotes. We used degenerate polymerase chain reaction (PCR) and Rapid Amplification of cDNA Ends (RACE) to clone and sequence the entire cDNAs of both the per and tim genes in Aedes albopictus (Skuse). We also used the 5′ end of the Ae. albopictus per cDNA to identify previously unannotated sequence coding for the N-terminal region of the PERIOD protein in Aedes aegypti L. We sequenced genomic DNA of one mosquito from each of three geographically distinct populations (New Jersey, Florida, and Brazil), and identified three introns in the per gene and eight introns in the tim gene. Phylogenetic analyses and comparison of functional domains support the orthology of the newly identified per and tim genes. Analysis of nonsynonymous to synonymous substitution rates indicates that both the per and tim genes have evolved under strong selective constraint subsequent to the divergence of Ae. albopictus and Ae. aegypti. Taken together, these results provide resources that can be used to investigate the molecular genetics of circadian phenotypes in Ae. albopictus and other culicids, to perform comparative analyses of insect circadian clock function, and also to conduct phylogeographic analyses using single-copy nuclear introns.

The gophertortoise tick, Amblyomma tuberculatum (Marx), is distributed throughout the southeastern United States, and its immature life stages have been reported to occasionally bite humans. Here we report detection of a novel spotted fever group (SFG) Rickettsia in A. tuberculatum ticks collected in the southern United States. Among questing ticks collected in Georgia, 10 pools of larvae were identified as gophertortoise ticks, A. tuberculatum. Each of these samples was positive for SFG Rickettsiae. The restriction fragment-length polymorphism profiles were identical to each other, but distinct from those of other rickettsiae previously found in Amblyomma spp. ticks. Partial genetic characterization of the novel agent was achieved by sequencing the 17 kDa, gltA, ompB, ompA, rpoB, and sca4 genes. Analysis of a concatenated tree of four genes (gltA, ompB, ompA, and sca4) demonstrates close relatedness of the detected Rickettsia to several SFG Rickettsia spp. The identical rickettsial DNA was detected in 50 and 70% of adult A. tuberculatum ticks from Mississippi and Florida, respectively. The results indicate wide distribution of a novel Rickettsia, capability for transovarial transmission, and high prevalence in tested tick populations.