Density-dependent intraspecific competition has been considered an important determinant of the dynamics of larval stages of Aedes aegypti. A model was published in 1984 providing a mathematical description of this density dependence, based on field data, that has since been widely used. This description, however, is based on the strong assumption that all mortality is density-dependent. We re-examine the data without this premise and find a reduced importance of density dependence, as well as a different functional form. Based on these discrepancies, we emphasize that the characterization of density dependence in the larval stages of Ae. aegypti should be based on a more complete dataset, and we use artificially generated data to explore how such additional information could help developing a better description of this density dependence. We review other empirical studies on larval competition, discuss the need for further dedicated studies, and provide a few simple guidelines for the design of such studies.

Cockroach suppression is fundamental to cockroach allergen mitigation in infested homes. The effects of various cockroach control strategies on cockroach populations and allergen concentration have not been examined in schools. This study was conducted to compare the effectiveness of integrated pest management (IPM) and conventional pest control in controlling German cockroach (Blattella germanica L.) infestations and concentrations of the cockroach allergen Bla g 1 in public school buildings. Two school districts included six schools that used conventional pest control and one district included seven schools that used IPM to control pests. Cockroach traps were deployed to assess the level of infestation, settled dust samples were collected in food service areas, classrooms, and other school areas, and the Bla g 1 allergen was quantified by ELISA. Both cockroach counts and Bla g 1 concentrations were dependent on the pest control approach, with highly significant differences between IPM-treated schools and conventionally treated schools in both the cockroach mean trap counts (0 versus 82.6 ± 17.3 cockroaches/trap/wk, respectively) and in the amount of Bla g 1 in dust samples (2.8 ± 0.3 versus 30.6 ± 3.4 U/g dust). Cockroaches and Bla g 1 were primarily associated with food preparation and food service areas and much less with classrooms and offices. Our data extend recent findings from studies in homes, showing that cockroach allergens can be reduced by cockroach elimination alone or by integrating several tactics including education, cleaning, and pest control. IPM is not only effective at controlling cockroaches but also can lead to long-term reductions in cockroach allergen concentrations, resulting in a healthier environment for students and school personnel.

Improving vector control remains a key goal in reducing the world's burden of infectious diseases. More cost-effective approaches to vector control are urgently needed, particularly because vaccines are unavailable and treatment is prohibitively expensive. The causative agent of American visceral leishmaniasis (AVL), Leishmania chagasi, Cunha and Chagas (Kinetoplastida: Trypanosomatidae), is transmitted between animal and human hosts by blood-feeding female sand flies attracted to mating aggregations formed on or above host animals by male-produced sex pheromones. Our results show the potential of using synthetic pheromones to control populations of Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae), the sand fly vector of one of the world's most important neglected diseases, AVL. We showed that a synthetic pheromone, (±) -9-methylger-macrene-B, produced from a low-cost plant intermediate, attracted females in the laboratory. By formulating dispensers that released this pheromone at a rate similar to that released by aggregating males, we were able to attract flies of both sexes to traps in the field. These dispensers worked equally well when deployed with mechanical light traps and inexpensive sticky traps. If deployed effectively, pheromone-based traps could be used to decrease AVL transmission rates through specific targeting and reduction of L. longipalpis populations. This is the first study to show attraction of a human disease-transmitting insect to a synthetic pheromone in the field, showing the general applicability of this novel approach for developing new tools for use in vector control.

This work studied the ultrastructure of the midgut cells of Cimex hemipterus Fabricius (Hemiptera: Cimicidae). The midgut of adult insects was analyzed on different days after a bloodmeal, and three anatomical regions with different digestive functions were apparent. In the anterior midgut, the digestive cells had many spherocrystals, lipid inclusions, and glycogen deposits, suggesting a role in water absorption, ion regulation, digestion, and storage of lipids and sugars. The digestive cells in the middle midgut contained secretory granules in the apical cytoplasm, lysosomes, and large amounts of rough endoplasmic reticulum, suggesting that this midgut region was active in digestive processes. The posterior midgut contained digestive cells with secretory vesicles, lysosomes, rough endoplasmic reticulum, and spherocrystals, suggesting digestion and ion/water absorption. Also, there was strong evidence that the posterior midgut may be the major site of nutrient absorption. The hematophagous heteropteran groups share many of these blood digestion mechanisms.

Hirstiella jimenezi new species is described from specimens found on the gecko Phyllodactylus bordai Taylor in the southern part of Mexico and also recorded from a single specimen of P. tuberculosus magnus Taylor. The female, male, deutonymph, and larva are described and illustrated. This is the first finding of Pterygosomatid mites on hosts of this genus.

Wolbachia pipientis Hertig and Wolbach (Rickettsiales: Rickettsiaceae) are intracellular α-proteobacteria that occur naturally in Aedes albopictus (Skuse) (Diptera: Culicidae) and numerous other invertebrates. These endosymbionts can invade host populations by manipulating host reproduction. Wolbachia infections have been shown to impart both costs and benefits to hosts in terms of development, survival, and fecundity. Here, we monitor intraspecific competition among independent cohorts of infected or uninfected larvae. Levels of competition are manipulated by varying initial larval densities and food levels. Although larval density is observed to have major impacts on immature survivorship, sex ratio of eclosing adults, and developmental rates, the Wolbachia infection status had minimal impact on male immatures and no effect on immature females under these experimental conditions. Female and male immatures were observed to respond differently to competitive pressure, with the functional relationships of females and males consistent with scramble and contest competition, respectively. The results are discussed in relation to the evolution of naturally occurring Wolbachia infections in Ae. albopictus (i.e., natural population replacement events) and public health strategies that propose the manipulation of Wolbachia infections in Ae. albopictus populations.

The interactions between predator diversity and primary consumer abundance can include direct effects and indirect, cascading effects. Understanding these effects on immature Anopheles mosquitoes is important in sub-Saharan Africa, where most cases of malaria occur. Aquatic predators and immature mosquitoes were collected from shallow pools of varying age previously excavated by brickmakers in the western highlands of Kenya. Path analysis showed an indirect negative effect of habitat age on An. gambiae (Giles, 1902) mediated by effects on predator diversity. Disturbance resets habitats to an earlier successional stage, diminishing predator diversity and increasing An. gambiae populations. The increase in vector abundance as a result of reduced predator diversity highlights the public health value in conserving native insect diversity.

Studies on the interactions of exotic species with their invaded environment are imperative in understanding their invasion biology. Larvae of container mosquitoes such as the invasive Aedes albopictus (Skuse) feed on microorganisms that subsist on allochthonous inputs like leaves. Ae. albopictus are vectors for many diseases including West Nile virus and are rapidly expanding their distribution in the United States. We tested the larval performance of Ae. albopictus at different larval densities in maple, oak, American elm, and persimmon. Survival was significantly lower and days to pupation were significantly higher with persimmon leaves compared with all others. In a follow-up experiment, we compared the performance of Ae. albopictus in different amounts of oak and persimmon and different ratios of persimmon oak. The linear model for the growth rate (defined by larval head width) showed a positive slope as the amount of oak leaves increased in oak treatment, but there was no significant slope for persimmon. In the persimmon oak combination, as the ratio of persimmon to oak increased, the growth rates of the larvae decreased. Lack of a significant slope for survival rate in combination with the results from the growth rate indicated that persimmon was a poor nutritional resource for Ae. albopictus.

In sexual organisms, the way in which gametes associate can greatly influence the maintenance of genetic variation, the structure of this variation in space, and ultimately organismal evolution. Based on patterns of genetic structure previously found, we explicitly tested whether adults of the sheep tick Ixodes ricinus pair according to their genetic relatedness. We sampled tick pairs from the vegetation in four natural populations and genotyped individual ticks at seven microsatellite loci. Based on this data, we observed highly significant assortative mating in two of the four locations, a pattern that could not be accounted for by a spatial autocorrelation in the distribution of related ticks. One explanation for these observations may be the existence of local host associations that develop independently in different populations. Assortative mating in I ricinus will have clear consequences for its population dynamics and, through processes of adaptation and transmission, may significantly alter the epidemiological patterns of the pathogens it carries, including the Lyme disease agent Borrelia burgdorferi s.l. Future tests will now be required to examine the mechanisms leading to this pattern and its epidemiological consequences.

The Rocky Mountain wood tick, Dermacentor andersoni Stiles, 1908, is of medical and veterinary importance because it can transmit pathogenic agents to humans, domestic livestock, and wildlife. The preferred attachment sites of D. andersoni adults and their ability to induce paralysis in hosts vary among populations, which may have a genetic basis. In this study, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analyses and DNA sequencing were used to determine the genetic variation in the 16S mitochondrial DNA gene of two D. andersoni populations from the Canadian prairies: Saskatchewan Landing Provincial Park, Saskatchewan, and Lethbridge, Alberta. Five haplotypes were detected in each population, but this was considerably lower than the 14 haplotypes reported in a previous study of a laboratory colony of D. andersoni originating from the Rocky Mountains in Montana. In addition, the Canadian populations did not share any haplotypes with the population from Montana. Differences in the genetic composition of the two Canadian prairie populations of D. andersoni compared with the montane population in the United States may have arisen through geographical isolation. These genetic differences between tick populations may also have important implications with respect to their ability to transmit pathogens to hosts. Further studies are needed to determine the extent of genetic variation and the vector potential of ticks from different populations throughout the range of D. andersoni in the United States and Canada.

Single-strand conformation polymorphism (SSCP) analysis was examined in a 303-bp region of the 16S and 12S mitochondrial rDNA genes to study haplotype frequencies among populations of Gulf Coast ticks collected from Refugio Co., TX, Payne Co., OK, and two sites in Osage Co., KS. Seven haplotypes were identified from the 16S rDNA gene fragment, whereas only two haplotypes were detected from the 12S fragment. Only the results from the 16S rDNA fragment are discussed. Haplotype diversity was greatest in Kansas (site 1), where three of the four haplotypes detected were unique to this site. All Gulf Coast tick populations shared the fourth haplotype. Two haplotypes were determined for Texas and Oklahoma populations, one of which appeared only in Texas, whereas the other was shared. Nei's haplotype diversity (h) indicated that the Texas population was relatively homogeneous (15%), whereas the remaining populations were heterogeneous (42–59%), although the Bonferroni confidence interval found no significant differences (P < 0.05). Nucleotide sequencing of the seven haplotypes and subsequent phylogenetic analysis using neighbor joining showed a monophyletic relationship among these haplotypes. One haplotype, shared by both Oklahoma and Kansas (site 2), was basal to the remaining haplotypes and formed a distinct clade. Two haplotypes, both from Kansas (site 1), formed a unique clade, whereas the remaining four haplotypes were unresolved polytomies.

Temperature is important for mosquito development and physiological response. Several genes of heat shock protein (HSP) families are known to be expressed in mosquitoes and may be crucial in responding to stress induced by elevated temperature. Suppression subtractive hybridization (SSH) was used to identify target transcripts to heat shock treatment in female Aedes aegypti. Subtraction was performed in both directions enriching for cDNAs differentially expressed between a non- heat shock control and heat shock treatment. Heat shock treatment of female Ae. aegypti was carried out for 1 h at 42°C. Clones from differentially expressed genes were evaluated by sequencing. Target transcripts up-regulated by heat shock included five different HSP gene families and 27 other genes, such as cytochrome c oxidase, serine-type endopeptidase, and glutamyl aminopeptidase. Additionally, some novel genes, cytoskeleton and ribosomal genes, were found to be differentially expressed, and three novel up-regulated sequences belonging to a low-abundance class of transcripts were obtained. Up-regulated/down-regulated transcripts from heat shock treatment were further confirmed and quantified by quantitative real-time polymerase chain reaction (PCR). High temperatures can alter the gene expression of a vector mosquito population, and further characterization of these differentially expressed genes will provide information useful in understanding the genetic response to heat shock treatment, which can be used to develop novel approaches to genetic control.

Heat shock genes are highly evolutionarily conserved and are expressed to varying degrees in all organisms in response to stress. Heat shock 70 (hsp70) genes have been well characterized in a number of organisms, most notably Drosophila melanogaster, but not as yet for any of the major arthropod-borne viral mosquito vectors. To identify hsp70 genes in the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae), basic local alignment searches of the Ae. aegypti genome were performed using D. melanogaster Hsp70 protein sequences as query. Two clusters of six previously unannotated AaHsp70 genes were identified and found to be organized into three pairs of nearly identical open reading frames, which mapped to two genomic scaffolds. Consistent with a designation as heat shock genes, no detectable level of expression of AaHsp70 genes was observed under normal rearing conditions (28°C), with robust expression observed with a heat shock of 37–39°C. Northern analysis showed heat-inducible expression of putative AaHsp70 genes at all life stages and in all tissues tested in a time- and temperature-dependent manner. Monitoring of AaHsp70 gene expression levels in field-caught Ae. aegypti may serve as a general marker for stress. In addition, promoter sequences from AaHsp70 genes may be used to control the expression of transgenes in an inducible manner.

Screening homes is an effective way of reducing house entry by mosquitoes. Here, we assess how important blocking the eaves is for reducing house entry by anopheline and culicine mosquitoes for houses that have screened doors and no windows. Twelve houses, with two screened doors and no windows, in which a single adult male slept, were included in a simple crossover design. In the first period, six houses were randomly selected and had the eaves blocked using a mixture of rubble and mortar; the other six were left with open eaves. Mosquitoes were sampled using CDC light traps from each house twice a week for 4 wk. Mosquito control activities and the number and type of domestic animals within the compound was recorded on each sampling occasion. Before beginning the second sampling period, homes with blocked eaves had them opened, and those with open eaves had them closed. Mosquitoes were then sampled from each house for a further 4 wk. When houses had their eaves closed, a three-fold reduction in Anopheles gambiae s.l. Giles caught indoors was observed. However, there was no reduction in total culicine numbers observed. This study demonstrates that the eaves are the major route by which An. gambiae enters houses. By contrast, culicine mosquitoes enter largely through doors and windows. Sealing the eave gap is an important method for reducing malaria transmission in homes where doors and windows are screened.

The aim of this study was to evaluate the fumigant and repellent activity of five essential oils (from eucalyptus, geranium, lavender, mint, and orange oil) and seven monoterpenes (eucalyptol, geraniol, limonene, linalool, menthone, linalyl acetate, and menthyl acetate) on first-instar nymphs of the bloodsucking bug Rhodnius prolixus Stahl (vector of Chagas disease in several Latin American countries). Fumigant activity was evaluated by exposing the nymphs to the vapors emitted by 100 ×l of essential oil or monoterpene in a closed recipient. The knockdown time 50% (KT50) for eucalyptus essential oil was 215.6 min (seven times less toxic than dichlorvos, a volatile organophosphorus insecticide used as a positive control). The remaining essential oils showed a poor fumigant activity: <50% of nymphs were knocked down after 540 min of exposure. The KT50 values for monoterpenes, expressed in minutes, were as follows: 117.2 (eucalyptol), 408.7 (linalool), 474.0 (menthone), and 484.2 (limonene). Eucalyptol was 3.5 times less toxic than dichlorvos. No affected nymphs were observed after 540 min of exposure to geraniol, linalyl acetate, or menthyl acetate. Repellency was quantified using a video tracking system. Two concentrations of essential oils or monoterpenes were studied (40 and 400 ×g/cm²). Only mint and lavender essential oils produced a light repellent effect at 400 ×g/cm². Geraniol and menthyl acetate produced a repellent effect at both tested concentrations and menthone only elicited an effect at 400 ×g/cm². In all cases, the repellent effect was lesser than that produced by the broad-spectrum insect repellent N,N-diethyl-3-methylbenzamide (DEET).

Pyrethrum is a natural mixture of six insecticidal esters, recognized for low mammalian toxicity and limited persistence in the environment. In this study, World Health Organization standard bioassays were used to evaluate the performance of pyrethrum against both susceptible and pyrethroid-resistant Anopheles gambiae s.s. The results showed that the intrinsic activity of pyrethrum was similar to that of permethrin but lower than that of deltamethrin against susceptible mosquitoes. However, pyrethrum was less affected by the presence of the kdr mutation than synthetic pyrethroids (with lower resistance ratios) and showed good knock-down effect, repellency, and blood-feeding inhibition against the pyrethroid-resistant strain. In laboratory condition, mosquito nets treated with 500–1,000 mg/m² (pyrethrum) remained effective, i.e., >80% mortality and/or >95% KD effect, for 9 mo. Conversely, the efficacy and residual activity of pyrethrum (Pynet 5% EC) on substrates was not conclusive, especially concerning mud, which is a porous substrate (mortality <80% after 3 mo at 2 g/m²). These findings suggested that pyrethrum may be a potential alternative candidate for the impregnation of mosquito nets and textiles in areas where resistance to pyrethroids has become problematic.

In Tunisia, the mosquito Culex pipiens shows various organophosphate resistance alleles at Ester and ace-1 loci. The characterization and the distribution pattern of these alleles were studied among 20 populations sampled from north to center of Tunisia. At the Ester locus, Ester⁴, Ester⁵, and Ester^B12 were present. A new esterase characterized by the same electrophoretic migration as esterase Al was identified: A13, encoded by EsterA¹³ allele. At the ace-1 locus, the presence of the ace-1^R, ace-1^D, and F290V mutated alleles was also detected. A large heterogeneity in allelic frequencies at Ester and ace-1 loci was observed among samples, with a high significant genotypic differentiation considering both loci (F_st = 0.077, P <10^-5), depicting variations of insecticide treatment intensity between areas. A comparison between populations collected in 1996 and 2005 showed an absence of significant resistance evolution. However, the high frequencies of resistance alleles in 2005 populations suggested that the selection pressures are still important in Tunisia. Strategies for resistance management are discussed in the context of the current knowledge of the Tunisian situation.

The ovicidal efficacy of two entomopathogenic hyphomycetes fungi—Metarhizium anisopliae variety acridum (M. an. ac.) Driver and Milner (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae variety anisopliae (M. an. an.) (Metschn.) Sorokin (Hypocreales: Clavicipitaceae)—was evaluated against eggs of three tick species (Acari: Ixodidae)—Hyalomma excavatum (Koch), Rhipicephalus (Boophilus) annulatus (Say), and Rhipicephalus sanguineus (Latereille)—by placing eggs, laid by surface-sterilized females, on conidia-impregnated filter paper. Although M. an. an. strains differed in their virulence to the tested ticks, they reduced the hatching percentages of eggs of all three tick species to 0–32% compared with 80–90% in the control eggs. The M. an. ac. strains were found highly virulent to H. excavatum and R. sanguineus eggs, reducing the hatching percentages to 2–6% but had no influence on hatching of R. annulatus eggs. Older tick eggs were more susceptible to fungal infection than newly laid ones. The effects of polar and nonpolar lipid fractions, extracted from the surface of tick eggs, on the development of conidia were tested. Both germination of M. an. an. conidia and formation of appressoria were stimulated by extracts from egg cuticles of all three tested tick species. However, the stimulating effect was lower when the conidia were exposed to lipids from relatively less susceptible R. annulatus eggs than when exposed to lipids from H. excavatum or R. sanguineus eggs. Unlike those of M. an. an., conidia of M. an. ac. exposed to such lipid extracts did not germinate and did not form appressoria.

Seven field strains of Culex pipiens pallens were evaluated in the summer of 2004 for their susceptibility to three insecticides. Larval bioassays were carried out in accordance with WHO mosquito susceptibility tests. Field samples showed resistance to dichlorvos and propoxur. Low levels of resistance to phoxim were detected compared with the laboratory-susceptible strain. Quantitative analysis showed that co-amplification of estα2 and estβ2 occurred in the seven field Cx. pipiens pallens. However, the levels of transcription of estα2/estβ2 genes in the field strains indicated a 15.9:1 ratio of estβ2 cDNA over estα2 cDNA. Such observed differential transcription of the estα2/estβ2 genes showed that the promoters regulating their expression are independent and of different strengths. Regression analysis of expression level of estα2/estβ2 gene and LC₅₀ value to insecticides showed a significant correlation between expression level of estα2 gene and LC₅₀ value to dichlorvos. However, correlation of estα2 gene and LC50 value to propoxur and phoxim, or estβ2 gene and three kinds of insecticides, was not significant.

We examined the potential of using native fish species in regulating mosquitoes in the floodplain of the Gambia River, the major source of mosquitoes in rural parts of The Gambia. Fishes and mosquito larvae were sampled along two 2.3-km-long transects, from the landward edge of the floodplain to the river from May to November 2005 to 2007. A semifield trial was used to test the predatory capacity of fish on mosquito larvae and the influence of fish chemical cues on oviposition. In the field, there was less chance of finding culicine larvae where Tilapia guineensis, the most common floodplain fish, were present; however, the presence of anophelines was not related to the presence or absence of any fish species. In semifield trials, both T. guineensis and Epiplatys spilargyreius were effective predators, removing all late-stage culicine and anopheline larvae within 1 d. Fewer culicines oviposited in sites with fish, suggesting that ovipositing culicine females avoid water with fish. In contrast, oviposition by anophelines was unaffected by fish. Our studies show that T. guineensis is a potential candidate for controlling mosquitoes in The Gambia.

Entomopathogenic fungi are commonly found in forested soils that provide tick habitat, and many species are pathogenic to Ixodes scapularis Say, the blacklegged tick. As a first step to developing effective biocontrol strategies, the objective of this study was to determine the best methods to isolate entomopathogenic fungal species from field-collected samples of soils and ticks from an Eastern deciduous forest where I. scapularis is common. Several methods were assessed: (1) soils, leaf litter, and ticks were plated on two types of media; (2) soils were assayed for entomopathogenic fungi using the Galleria bait method; (3) DNA from internal transcribed spacer (ITS) regions of the nuclear ribosomal repeat was extracted from pure cultures obtained from soils, Galleria, and ticks and was amplified and sequenced; and (4) DNA was extracted directly from ticks, amplified, and sequenced. We conclude that (1) ticks encounter potentially entomopathogenic fungi more often in soil than in leaf litter, (2) many species of potentially entomopathogenic fungi found in the soil can readily be cultured, (3) the Galleria bait method is a sufficiently efficient method for isolation of these fungi from soils, and (4) although DNA extraction from ticks was not possible in this study because of small sample size, DNA extraction from fungi isolated from soils and from ticks was successful and provided clean sequences in 100 and 73% of samples, respectively. A combination of the above methods is clearly necessary for optimal characterization of entomopathogenic fungi associated with ticks in the environment.

The cost and effectiveness of two bed bug (Cimex lectularius L.) integrated pest management (IPM) programs were evaluated for 10 wk. Sixteen bed bug-infested apartments were chosen from a high-rise low-income apartment building. The apartments were randomly divided into two treatment groups: diatomaceous earth dust-based IPM (D-IPM) and chlorfenapyr spray-based IPM (S-IPM). The initial median (minimum, maximum) bed bug counts (by visual inspection) of the two treatment groups were 73.5 (10, 352) and 77 (18, 3025), respectively. A seminar and an educational brochure were delivered to residents and staff. It was followed by installing encasements on mattresses and box springs and applying hot steam to bed bug-infested areas in all 16 apartments. Diatomaceous earth dust (Mother Earth-D) was applied in the D-IPM group 2 d after steaming. In addition, bed bug-intercepting devices were installed under legs of infested beds or sofas or chairs to intercept bed bugs. The S-IPM group only received 0.5% chlorfenapyr spray (Phantom) after the nonchemical treatments. All apartments were monitored bi-weekly and retreated when necessary. After 10 wk, bed bugs were eradicated from 50% of the apartments in each group. Bed bug count reduction (mean ± SEM) was 97.6 ± 1.6 and 89.7 ± 7.3% in the D-IPM and S-IPM groups, respectively. Mean treatment costs in the 10-wk period were $463 and $482 per apartment in the D-IPM and S-IPM groups, respectively. Bed bug interceptors trapped an average of 219 ± 135 bed bugs per apartment in 10 wk. The interceptors contributed to the IPM program efficacy and were much more effective than visual inspections in estimating bed bug numbers and determining the existence of bed bug infestations.

We demonstrate that the addition of bed bug, Cimex lectularius,, alarm pheromone to desiccant formulations greatly enhances their effectiveness during short-term exposure. Two desiccant formulations, diatomaceous earth (DE) and Dri-die (silica gel), were applied at the label rate with and without bed bug alarm pheromone components, (E)-2-hexenal, (E)-2-octenal, and a (E)-2-hexenal: (E)-2-octenal blend. First-instar nymphs and adult females were subjected to 10-min exposures, and water loss rates were used to evaluate the response. Optimal effectiveness was achieved with a pheromone concentration of 0.01 M. With Dri-die alone, the water loss was 21% higher than in untreated controls, and water loss increased nearly two times with (E)-2-hexenal and (E)-2-octenal and three times with the (E)-2-hexenal: (E)-2-octenal blend. This shortened survival of first-instar nymphs from 4 to 1 d, with a similar reduction noted in adult females. DE was effective only if supplemented with pheromone, resulting in a 50% increase in water loss over controls with the (E)-2-hexenal: (E)-2-octenal blend, and a survival decrease from 4 to 2 d in first-instar nymphs. Consistently, the addition of the pheromone blend to desiccant dust was more effective than adding either component by itself or by using Dri-die or DE alone. Based on observations in a small microhabitat, the addition of alarm pheromone components prompted bed bugs to leave their protective harborages and to move through the desiccant, improving the use of desiccants for control. We concluded that short exposure to Dri-die is a more effective treatment against bed bugs than DE and that the effectiveness of the desiccants can be further enhanced by incorporation of alarm pheromone. Presumably, the addition of alarm pheromone elevates excited crawling activity, thereby promoting cuticular changes that increase water loss.

Using the polymerase chain reaction (PCR)-select subtractive cDNA hybridization technique, 18 different genes were isolated from a permethrin-treated versus acetone-treated Aedes aegypti subtractive library. Quantitative PCR (QPCR) results showed that 8 of the 18 gene's transcriptional levels in permethrin-treated Ae. aegypti were at least two-fold higher (ranging from 2.6 ± 0.5 to 4.8 ± 0.2) than that in acetone-treated Ae. aegypti. These eight genes include three functionally known genes (cytochrome c oxidase subunit III, NADH2 dehydrogenase, deltamethrin resistance associated protein), three functionally unknown genes (Ae. aegypti putative 16.9-kDa secreted protein, Anopheles gambiae ENSANGP00000019508, Cryptococcus neoformans hypothetical protein CNE05340), and two novel genes. Transcriptional levels for 11 of the 18 genes were induced significantly higher by permethrin than by fipronil (P < 0.05). Our results suggest that subtractive cDNA hybridization and QPCR are powerful techniques to identify differentially expressed genes in response to pesticide treatment.

Plague, the disease caused by the bacterium Yersinia pestis, can have devastating impacts on black-tailed prairie dogs (Cynomys ludovicianus Ord). Other mammal hosts living on prairie dog colonies may be important in the transmission and maintenance of plague. We examined the flea populations of northern grasshopper mice (Onychomys leucogaster Wied) before, during, and after plague epizootics in northern Colorado and studied the influence of host and environmental factors on flea abundance patterns. Grasshopper mice were frequently infested with high numbers of fleas, most commonly Pleochaetis exilis Jordan and Thrassis fotus Jordan. Flea loads changed in response to both environmental temperature and rainfall. After plague-induced prairie dog die-offs, flea loads and likelihood of infestation were unchanged for P. exilis, but T. fotus loads declined.

The human skin contacts molecules from house dust mites that are ubiquitous in many environments. These mite-derived molecules may penetrate the skin epidermis and dermis and contact microvascular endothelial cells and influence their function. The purpose of this study was to determine the response of normal human dermal microvascular endothelial cells to extracts of the dust mites, Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei with and without endotoxin (lipopolysaccharide). Endothelial cells were stimulated with mite extracts and the expression of surface molecules and the secretion of cytokines were measured in the absence and presence of polymyxin B to bind endotoxin. All three mite extracts stimulated endothelial cells to express intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin and to secrete interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP-1), and granulocyte/macrophage colony stimulating factor (GM-CSF). Euroglyphus maynei-induced expression of all the cell surface molecules was not inhibited when the endotoxin activity in the mite extract was inhibited. In contrast, endothelial cells challenged with D. farinae or D. pteronyssinus extract depleted of endotoxin activity expressed only constitutive levels of ICAM-1, VCAM-1, and E-selectin. D. farinae and E. maynei extracts depleted of endotoxin activity still induced secretion of IL-8 and MCP-1 but at reduced levels. Only constitutive amounts of IL-6, G-CSF, and GM-CSF were secreted in response to any of the endotoxin-depleted mite extracts. Extracts of D. farinae, D. pteronyssinus, and E. maynei contain both endotoxins and other molecules that can stimulate expression of cell adhesion molecules and chemokine receptors and the secretion of cytokines by normal human microvascular endothelial cells.

It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis.

The recent spread of Bluetongue disease in northwestern Europe has indicated the ability of Palaearctic Culicoides species to vector the disease. Because the different midge species vary in their ability to harbor and transmit the Bluetongue virus, quick and reliable identification is necessary to resolve the species composition of midge communities, both adult and larval, at any place at any given time point. Given that morphological identification of Culicoides species is problematic, we developed three multiplex polymerase chain reaction (PCR) assays that facilitate high-throughput analysis of midge specimens. One assay distinguishes between species of the so-called Culicoides obsoletus s.l. complex (including C. dewulfi), whereas two assays facilitate differentiation of species of the Culicoides pulicaris s.l. complex. These assays yield two PCR products: one species-specific and one generic band. We show the application of the assays in the analysis of Culicoides larvae from three different farms in northeast Scotland.

Biting midges in the genus Culicoides (Diptera: Ceratopogonidae) were collected near sunset by direct aspiration from sheep in northeastern Spain to determine species-specific biting rates and crepuscular activity. Midges were also collected by UV-baited light traps and CO₂-baited traps over the same period to compare species diversity and abundance using these common surveillance methods to actual sheep attack rates. Culicoides aspirated from sheep included C. obsoletus, C. parroti, C. scoticus, C. punctatus, and C. imicola. Peak host-seeking activity during the time period examined for the two most commonly collected species (C. obsoletus and C. parroti) occurred just before sunset and activity ceased within 1 h after sunset. Host attack rates near sunset averaged 0.9 midges/min for both species with maximum attack rates of 3/min for C. obsoletus and 4/min for C. parroti. For both species, ≈35% of midges collected from the sheep were engorged, giving a maximum biting rate of 1.1/min for C. obsoletus and 1.5/min for C. parroti. Traps baited with CO₂ collected fewer midges of each species relative to other collection methods. Traps baited with UV light provided a good indication of species richness but significantly underestimated the host attack rate of C. obsoletus and C. parroti while overestimating the host attack rate of C. imicola. Animal-baited collecting is critical to interpret the epidemiological significance of light trap collections used for surveillance of the midge vectors of bluetongue virus and African horse sickness virus.

Dermacentor albipictus (Packard) is a North American tick that feeds on cervids and livestock. It is a suspected vector of anaplasmosis in cattle, but its microbial flora and vector potential remain underevaluated. We screened D. albipictus ticks collected from Minnesota white-tailed deer (Odocoileus virginianus) for bacteria of the genera Anaplasma, Ehrlichia, Francisella, and Rickettsia using polymerase chain reaction (PCR) gene amplification and sequence analyses. We detected Anaplasma phagocytophilum and Francisella-like endosymbionts (FLEs) in nymphal and adult ticks of both sexes at 45 and 94% prevalences, respectively. The A. phagocytophilum and FLEs were transovarially transmitted to F₁ larvae by individual ticks at efficiencies of 10–40 and 95–100%, respectively. The FLEs were transovarially transmitted to F₂ larvae obtained as progeny of adults from F₁ larval ticks reared to maturity on a calf, but A. phagocytophilum were not. Based on PCR and tissue culture inoculation assays, A. phagocytophilum and FLEs were not transmitted to the calf. The amplified FLE 16S rRNA gene sequences were identical to that of an FLE detected in a D. albipictus from Texas, whereas those of the A. phagocytophilum were nearly identical to those of probable human-nonpathogenic A. phagocytophilum WI-1 and WI-2 variants detected in white-tailed deer from central Wisconsin. However, the D. albipictus A. phagocytophilum sequences differed from that of the nonpathogenic A. phagocytophilum variant-1 associated with Ixodes scapularis ticks and white-tailed deer as well as that of the human-pathogenic A. phagocytophilum ha variant associated with I. scapularis and the white-footed mouse, Peromyscus leucopus. The transovarial transmission of A. phagocytophilum variants in Dermacentor ticks suggests that maintenance of A. phagocytophilum in nature may not be solely dependent on horizontal transmission.

A 4-yr (2002–2006) entomological study was carried out in Kurnool district, Andhra Pradesh state, south India, to identify the mosquito vectors of Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV). In total, 37,139 female mosquitoes belonging five genera and 18 species resting on vegetation were collected in villages and periurban areas at dusk. Mosquito species composition and pattern of JEV infection in mosquitoes varied in periurban and rural areas. In periurban area, Culex gelidus Theobald was abundant, msking up 49.7% of total catch followed by Culex tritaeniorhynchus Giles (44.5%). In rural area, Cx. tritaeniorhynchus was predominant, making up 78.9% of total catch followed by Culex quinquefasciatus Say (10.8%), Anopheles subpictus Grassi (7.1%), and Cx. gelidus (1.1%). In light trap collections, Cx. gelidus and Cx. tritaeniorhynchus predominated in periurban and rural areas, respectively. Of 50,145 mosquitoes screened JEV isolations were made only from Cx. gelidus and Cx. tritaeniorhynchus. Based on high abundance and frequent JEV isolation, Cx. tritaeniorhynchus was found to be the principal vector in both areas, whereas Cx. gelidus plays a secondary vector role in periurban areas only.

Mosquitoes collected during 2003, 2004, and 2005 in Alberta, Canada, were screened for the presence of a wide range of arboviruses by reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acid extracts from mosquito slurries were amplified using universal primers designed to detect viruses belonging to the Flavivirus genus of the Flaviviridae family and California and Bunyamwera serogroups of the Bunyavirus genus within the Bunyaviridae family. Species-specific detection of Western equine encephalitis virus and Eastern equine encephalitis virus was also performed. Amplified products were analyzed, and the viral target was identified by sequencing. Of the 418 pools tested, 3 pools contained Cache Valley virus belonging to Bunyaviridae and 103 pools were positive for a previously undescribed flaviviral sequence that was most similar to Kamiti River virus. These data suggest that nucleic acid amplification using broadly reactive primers can be adopted for arbovirus surveillance in mosquito populations, and this approach has the potential to detect both previously recognized and novel viruses.

Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose- 6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donouam-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the “Hyper” gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

There is very little information concerning carrion fly population genetic structure. We generated amplified fragment length polymorphism (AFLP) profiles for the common blowfly, Phormia regina (Meigen), from sites spanning the contiguous United States. Analysis of molecular variance (AMOVA) based on 232 loci found significant variation (Φ_SC = 23%) among discrete samples (those collected at a bait in one location over a short period of time). Samples collected in the same location but at different times were also distinct. When samples were pooled into geographic regions (east, central, west), the variation was negligible (Φ_CT = 0%). A Mantel test found only a very weak correlation between individual genetic and geographic distances. Relative relatedness coefficients based on shared allele proportions indicated individual samples were likely to contain close relatives. P. regina arriving at an individual carcass typically represent a nonrandom sample of the population despite a lack of geographic structure. A female blow fly produces hundreds of offspring at one time; therefore, newly emerged siblings may respond in concert to an odor plume. These results may be of interest to forensic entomologists, many of whom use a laboratory colony founded from a small sample for the growth studies that support casework. Discrepancies between published growth curves may reflect such random differences in the founding individuals.

The most common application of forensic entomology involves estimating a portion of the postmortem interval (PMI), which usually assumes that blow flies (Diptera: Calliphoridae) do not oviposit nocturnally. Research objectives were to (1) investigate blow fly nocturnal oviposition in relation to sunrise and sunset in Michigan; (2) evaluate abiotic variables postulated to affect blow fly oviposition; and (3) conduct laboratory experiments testing blow fly activity under complete darkness. In 2006, nocturnal oviposition was evaluated in relation to sunset by exposing pigs to fly colonization at 1-h intervals, beginning 2 h before and ending 2 h after sunset. This test was replicated in 2007; however, replicate pigs were placed in the field 2 h after sunset, and hourly observations were made into the following morning. Oviposition was never observed at night. In a laboratory experiment, Lucilia sericata (Meigen), never oviposited on liver hanging above or placed directly on the ground in a completely dark room, Another dark room laboratory study documented that adult flies launched into the air could not fly. This study documents that the probability of nocturnal oviposition on pig carcasses in Michigan was extremely low to nonexistent. These results should be considered when estimating a portion of the PMI in forensic entomological investigations.

Environmental geospatial data and adult and larval mosquito collection data for up to 106 sites throughout the Republic of Korea (ROK) were used to develop ecological niche models (ENMs) of the potential geographic distribution for eight anopheline species known to occur there. The areas predicted suitable for the Hyrcanus Group species were the most extensive for Anopheles sinensis Wiedemann, An. kleini Rueda, An. belenrae Rueda, and An. pullus Yamada, intermediate for An. sineroides Yamada, and the most restricted for An. lesteri Baisas and Hu and the non-Hyrcanus Group species An. koreicus Yamada and Watanabe and An. lindesayi Yamada. The relative vectorial importance of these species is unknown, and all, except An. koreicus and An. lindesayi, are predicted to occur widely in the northwest of the ROK where malaria transmission has been sporadic since its resurgence in 1993. Our ENMs suggest that it is unlikely that An. koreicus and An. lindesayi are vectors, but we do not document consistent geographic differentiation that might incriminate any of the other species as vectors. Because all species are predicted to occur in North Korea, we also cannot reject the hypothesis that malaria infected mosquitoes from North Korea may have been the cause of the resurgence of malaria in the ROK. Ecological differentiation of the eight species is inferred from collection locations and 34 environmental layers based on remote sensing and global climatic averages. Interspecific differences were noted, and characterizing mosquito habitats by ground-based and remote sensing methods is proposed.

Leishmaniasis has been of concern to the U.S. military and has re-emerged in importance because of recent deployments to the Middle East. We conducted a retrospective probabilistic risk assessment for military personnel potentially exposed to insecticides during the “Leishmaniasis Control Plan” (LCP) undertaken in 2003 at Tallil Air Base, Iraq. We estimated acute and subchronic risks from resmethrin, malathion, piperonyl butoxide (PBO), and pyrethrins applied using a truck-mounted ultra-low-volume (ULV) sprayer and lambda-cyhalothrin, cyfluthrin, bifenthrin, chlorpyrifos, and cypermethrin used for residual sprays. We used the risk quotient (RQ) method for our risk assessment (estimated environmental exposure/toxic endpoint) and set the RQ level of concern (LOC) at 1.0. Acute RQs for truck-mounted ULV and residual sprays ranged from 0.00007 to 33.3 at the 95th percentile. Acute exposure to lambda-cyhalothrin, bifenthrin, and chlorpyrifos exceeded the RQ LOC. Subchronic RQs for truck-mounted ULV and residual sprays ranged from 0.00008 to 32.8 at the 95th percentile. Subchronic exposures to lambda-cyhalothrin and chlorpyrifos exceeded the LOC. However, estimated exposures to lambda-cyhalothrin, bifenthrin, and chlorpyrifos did not exceed their respective no observed adverse effect levels.

Fleas occur as ectoparasites of vertebrates around the world. The obligate intracellular bacterium Rickettsia felis has been detected globally in several flea species, causing a murine-typhus like disease in humans. In this study, a total of 150 hedgehog fleas (Archaeopsylla erinacei Bouché) were collected from 18 hedgehogs coming from four locations in southern Germany for the detection of R. felis. Individual DNA extracts were tested with polymerase chain reaction (PCR) for the amplification of the rickettsial ompB, gltA, ompA, and 16S rRNA genes. A total of 144 samples (96%) were positive using ompB PCR. Sequencing and phylogenetic analysis showed an organism very closely related to R. felis with 96% similarity. These results provided evidence that hedgehog fleas in Germany may be nearly 100% infected with a rickettsial species closely related to R. felis. Further studies are needed for its molecular and pathogenetic characterization. The hedgehog as a potential reservoir for the emergent pathogen R. felis or closely related strains also needs further study.

The main vectors of Chagas disease in Ecuador are Triatoma dimidiata and Rhodnius ecuadoriensis. The latter species occupies domestic and peridomestic habitats, as well as sylvatic ecotopes—particularly associated with Phytelephas aequatorialis palm trees—in the western coastal region of Ecuador. In the southern highlands, however, such palm tree habitats are uncommon, and sylvatic populations of R. ecuadoriensis have not previously been reported to date. This study was carried out in five rural communities in Loja Province in southern Ecuador, where manual triatomine searches were conducted in various sylvatic habitats. A total of 81 squirrel nests (Sciurus stramineus) and >200 bird nests and other habitats were searched. One hundred three R. ecuadoriensis individuals were found in 11 squirrel nests (infestation index = 13.6%, density = 2 bugs per nest searched, crowding = 9.5 bugs per infested nest, colonization index = 72.7% of infested nests with nymphs). No triatomines were found in bird nests or other sylvatic habitats. The presence of sylvatic R. ecuadoriensis in the southern highlands of Ecuador has important implications for the long-term control of Chagas disease in the region because of the possibility of reinfestation of dwellings after insecticide-based control interventions.

In this article, we present the results of a study about mites associated with bats collected in the State of Pernambuco, northeast Brazil. Of the 331 bats collected, 23 were found naturally infested by mites, corresponding to an overall prevalence of 6.9% (95% confidence interval: 4.5–10.2). Four mite species were collected from seven bat species. The following mite-bat associations were recorded: Periglischrus acutisternus Machado-Allison on Phyllostomus discolor Wagner; Periglischrus ojastii Oudemans on Carollia perspicillata L.; Periglischrus iheringi Oudemans on Artibeus lituratus (Olfers), Artibeus planirostris Spix, Platyrrhinus lineatus (E. Geoffroy), and Sturnira lilium (E. Geoffroy); and Spelaeorhynchus praecursor Neumann on C. perspicillata. This study definitively confirms the presence of S. praecursor in Pernambuco and records for the first time the presence of three spinturnicid species (i.e., P. acutisternus, P. ojastii, and P. iheringi) in northeast Brazil.