Dengue viruses, primarily transmitted by the mosquito Aedes aegypti (L.), affect an estimated 50–100 million people yearly. Traditional approaches to control mosquito population numbers, such as the use of pesticides, have had only limited success. Atypical mosquito behavior may be one reason why current vector control efforts have been less efficacious than expected. In Puerto Rico, for example, adult Ae. aegypti have been observed emerging from septic tanks. Interestingly, adults emerging from septic tanks are larger on average than adults collected from surface containers. To determine whether adults colonizing septic tanks constitute a separate Ae. aegypti population, we used 12 previously validated microsatellite loci to examine adult mosquitoes collected from both septic tanks and surface containers, but found no evidence to suggest genetic differentiation. Size differences between septic tank and surface mosquitoes were reduced when nutrient levels were held constant across experimental groups. Despite the absence of evidence suggesting a genetic difference between experimental groups in this study, Ae. aegypti emerging from septic tanks may still represent a more dangerous phenotype and should be given special consideration when developing vector control programs and designing public health interventions in the future.

Sand flies are small blood feeding dipterans that are primary vectors of numerous human and livestock pathogens. Control efforts are often complex and multidimensional. A fundamental step in the development and implementation of any integrated pest management (IPM) program is the refining of effective surveillance and identification techniques. Before this study, the presence of two species, Lutzomyia shannoni (Dyar) and Lutzomyia vexator (Coquillett) became known in Kentucky and surrounding states. To understand the spatial distribution of these species across heterogeneous landscapes in this region, trapping was conducted at random locations in previously designated habitats. Although a lack of clear data prevented any conclusions to be drawn for the preferences of L. vexator, the species of greater medical and veterinary importance, L. shannoni, was documented to show a strong preference for habitats along the wooded edges of pasture lands. In the region of study, these lands are frequently used in the production of beef cattle and other livestock, reinforcing the veterinary significance of the findings. Because of their familiarity to vector control officials and operators, mosquito species compositions were compared with the recorded abundance of L. shannoni across the varied habitats. The linking of this information should prove useful to management plans for the phlebotomine, if warranted in the future.

In criminal and civil legal investigations the forensic entomologist usually assists in providing an estimate of the postmortem interval, which closely couples with the time or period of insect activity. A minimum period of insect activity is often interpreted and estimated by dipteran larval developmental age of the oldest specimens collected at a crime scene and /or autopsy. In entomological evidence protocols investigators are recommended to search a 2–10 m perimeter area for the oldest larvae that may have begun to disperse away from the body for burial and pupation. In this study, we described a case of a large aggregate (>90% larvae) en masse postfeeding dispersal of blow fly larvae away from replicate swine carcasses serving as models of human decomposition. Larval dispersal was evaluated for a spring and a summer trial, with en masse characteristics only occurring during the latter. This en masse dispersal occurred in five out of six replicate carcasses and masses moved from 2 to 26 m away. These data and observations suggest the importance of performing searches >10 m from human remains for entomological evidence at crime scenes. Ry missing the oldest larvae at a crime scene, interpretation of entomological evidence can be compromised and erroneous. Based on these data and observations we recommend the crime scene investigators and researchers consider increasing the search radius around crime scene remains to increase the likelihood that the oldest larvae have been collected for analysis.

Nootkatone is a component of grapefruit oil that is toxic to the disease-vectoring tick, Ixodes scapularis Say, but unfortunately causes phytotoxicity to treated plants and has a short residual activity due to volatility. We prepared a lignin-encapsulated nootkatone formulation to compare with a previously used emulsifiable formulation for volatility, plant phytotoxicity, and toxicity to unfed nymphs of I. scapularis. Volatility of nootkatone was measured directly by trapping nootkatone vapor in a closed system and indirectly by measuring nootkatone residue on treated filter paper after exposure to simulated sunlight (Xenon). After 24 h in the closed system, traps collected only 15% of the nootkatone applied as the encapsulated formulation compared with 40% applied as the emulsifiable formulation. After a 1-h light exposure, the encapsulated formulation retained 92% of the nootkatone concentration compared with only 26% retained by the emulsifiable formulation. For plant phytotoxicity, cabbage, Brassica oleracea L., leaves treated with the encapsulated formulation expressed less necrosis, retaining greater leaf weight compared with leaves treated with the emusifiable formulation. The nootkatone in the emulsifiable formulation was absorbed by cabbage and oat, Avena sativa L., plants (41 and 60% recovered 2 h after application, respectively), as opposed to 100% recovery from the plants treated with encapsulated nootkatone. Using a treated vial technique, encapsulated nootkatone was significantly more toxic to I. scapularis nymphs (LC₅₀ = 20 ng/cm²) compared with toxicity of the emulsifiable formulation (LC₅₀ = 35 ng/cm²). Thus, the encapsulation of nootkatone improved toxicity for tick control, reduced nootkatone volatility, and reduced plant phytotoxicity.

The effect of Musca domestica salivary gland hypertrophy virus (MdSGHV) on selected fitness parameters of stable flies, Stomoxys calcitrans (L.), was examined in the laboratory. Virusinjected stable flies of both genders suffered substantially higher mortality than control flies. By day 9, female mortality was 59.3 ± 10.1% in the virus group compared with 23.7 ± 3.7% in the controls; mortality in virus-injected males was 78.1 ± 3.1% compared with 33.3 ± 9.3% for controls. Fecundity of control flies on days 6–9 was 49–54 eggs deposited per live female per day (total, 8,996 eggs deposited), whereas virus-injected flies produced four to five eggs per female on days 6–7 and less then one egg per female per day thereafter (total, 251 eggs). Fecal spot deposition by virus-injected flies was comparable to controls initially but decreased to ≈50% of control levels by day 4 after injection; infected flies produced only 26% as many fecal spots as healthy flies on days 6 and 7. None of the virus-injected stable flies developed symptoms of salivary gland hypertrophy. Quantitative real-time polymerase chain reaction demonstrated virus replication in injected stable flies, with increasing titers of virus genome copies from one to four days after injection. MdSGHV in stable flies displayed tissue tropism similar to that observed in house fly hosts, with higher viral copy numbers in fat body and salivary glands compared with ovaries. Virus titers were ≈2 orders of magnitude higher in house fly than in stable fly hosts, and this difference was probably due to the absence of salivary gland hypertrophy in the latter species.

Stormwater treatment systems (STS) intended to mitigate the potentially negative public health and environmental impact caused by urban runoff are highly conducive to mosquito production. Thirty-one STS, 15 aboveground extended detention basins (EDBs) and 16 proprietary belowground systems newly installed along State Route 125 in San Diego County, CA, were inspected monthly between July 2008 and June 2009 for presence of standing water and mosquitoes. During the 12-mo study, standing water was observed in 66% of the 180 total inspections to EDBs and at least once in each of the 15 basins, whereas belowground systems held water year-round in permanent-water sumps. With the exception of one EDB, immature mosquitoes were observed in all STS, during every month of the year in EDBs and all months except December in belowground systems. Cumulatively, mosquitoes were noted in 44% of the 372 total site inspections, with a nearly equal number of positive observations from EDBs and belowground systems. Four species were identified from EDBs, Culex tarsalis Coquillett, Cx. quinquefasciatus Say, Anopheles hermsi Barr and Guptavanij, and Culiseta incidens (Thompson). Cx. quinquefasciatus was the sole species identified from belowground systems. Results derived from this study provide additional evidence for mosquito production in STS because of structural design or persistent inflows, or both, of dry-weather urban runoff. Interagency collaboration is needed to ensure that STS are designed and maintained in a way that minimizes their potential to produce mosquitoes that can negatively affect public health.

Reducing populations of phlebotomine sand flies in areas prevalent for human leishmaniases is of ongoing importance to United States military operations and civilian populations in endemic regions. However, not enough is known regarding the efficacy of Department of Defense-approved pesticides and equipment against sand flies; specifically, the potential for ultra-low volume (ULV) pesticide applications to control Old World sand fly vectors. In this study we examine two sprayers, the Terminator ULV and the Grizzly ULV, with UV-labeled Duet and Fyfanon in four combinations against caged Phlebotomus duboscqi (Neveu-Lemaire) and wild sand fly populations in a natural environment in western Kenya. All equipment and Fyfanon have United States military National Stock Numbers and both pesticides are registered with the United States Environmental Protection Agency. Caged sand flies were reared from local P. duboscqi and the area has long been studied because of high incidences of human cutaneous and visceral Leishmania. Patterns of mortality across grids of caged sand flies showed greater efficacy from the Grizzly ULV regardless of chemical. The Terminator ULV performed well with Duet but with a less uniform and overall lower rate of mortality across the spray grid. Sampling of wild populations before and after treatments suggested local population suppression from ULV treatments, as well as a possible repellent effect in nearby untreated areas. Surprisingly, ULV active ingredient deposition inferred from patterns of UV-labeled droplets captured on cotton ribbons adjacent to sand fly cages in spray plots did not match patterns of mortality. We discuss the implications of this study, the first of its kind, for future military preventive medicine activities, including relative performance costs and benefits of larger or smaller sprayers, and the relative stability of ULV-induced mortality patterns in varied or sub-optimal conditions.

The present work was designed to evaluate the toxicity of various synthesized aromatic amides that are analogs of N,N-diethyl-2-phenylacetamide, a well known insect repellent. The toxicity profile of these compounds was compared with N,N-diethyl-2-phenylacetamide as well as other registered insect repellents namely N,N-diethyl-3-methyl benzamide and N,N-diethylbenzamide. The primary skin irritation index values of the compounds, dermal toxicity of the chemicals and acute oral toxicity data to assess the safety of the synthesized aromatic amides are reported in this paper. Results of hematological and biochemical studies of these analogues are reported and discussed.

We analyzed the variability of susceptibility to deltamethrin in putatively susceptible Triatoma infestans Klug (Hemiptera: Reduviidae), and evaluated the sample size implications on the hypotheses used in the current World Health Organization protocol for the measure of insecticide resistance in Triatominae. Following the protocol, using topical application of deltamethrin to unfed first instar nymphs of T. infestans, we found that susceptibility showed significant differences between offspring from different females, a significant association with female age, and significant interaction female X female age. Considering individual female data, three patterns of nymphal mortality were identified: one showed a strong positive relation between nymphal mortality and their mother's age, another showed high mortality with low variability and the third showed intermediate mortality with high variability along female age. The analysis suggests revision of the World Health Organization protocol for resistance detection in Triatominae, not only to take into consideration the sources of variation in susceptibility, but also the effects of sample size in relation to the significance and power probabilities of the test.

The 0.5% ivermectin topical cream formulation was not directly ovicidal to treated eggs of head lice, as hatchability was not decreased. Nevertheless, the percent of hatched lice from treated eggs that took a blood meal significantly decreased (80–95%) compared with lice that hatched from untreated eggs and all treated lice died within 48 h of hatching, including those that fed. Dilutions of ivermectin formulation of 0.15 and 0.2 µg/ml, which were topically applied to 0–8 d old eggs, were not lethal to lice at 24 h posteclosion. However, 9 and 16% less lice fed when hatched from these treated eggs, respectively. Total [³H] inulin ingested by untreated first instars significantly increased over a 48 h feeding interval but was significantly less in instars that hatched from eggs receiving the 0.15 (36% less) and 0.2 (55% less) µg/ml ivermectin treatments compared with placebo. The reduced feeding that occurred after the 0.15 and 0.2 µg/ml ivermectin treatments occurred in the absence of mortality and suggests a unique mode of action of ivermectin on feeding that is separate from the mode of action of ivermectin leading to mortality. Failure of hatched instars to take a blood meal after egg treatments with formulated ivermectin is likely responsible for its action as a posteclosion nymphicide.

Evolutionary theory predicts that virulence of parasites for mobile vector insects will be low for natural parasite-host associations that have coevolved. I determined virulence of the malaria parasite of lizards, Plasmodium mexicanum, for its vectors, two species of sand fly (Diptera: Psychodidae), Lutzomyia vexator (Coquillett 1907) and Lutzomyia stewarti (Mangabeira Fo & Galindo 1944), by measuring several life history traits. Developmental rate from egg to eclosion differed for the two species when noninfected. For both sand fly species, developmental rate for each stage (egg to larval hatching, larval period, pupal period) and life span were not altered by infection. Infected sand flies, however, produced fewer eggs. This reduction in fecundity may be a result of lower quality of the blood meal taken from infected lizards (lower concentration of hemoglobin). This report is the first measure of virulence of Plasmodium for an insect vector other than a mosquito and concords with both expectations of theory and previous studies on natural parasite-host associations that revealed low virulence.

A previously validated, spatially explicit, agent-based model of arbovirus transmission among vector mosquitoes and avian hosts is used to explore the effectiveness of different insecticide control strategies for reducing rates of epizootic transmission and mosquito infection. Mosquito control within the model reduces infection rates among hosts and vectors but generally lengthens the transmission season. Insecticide application focused at oviposition sites is found to be as effective as control applied over the entire model landscape. The findings also indicate that optimum use of insecticide resources would first target areas in which bird activity is collocated with vector mosquito breeding sites, and then, if resources permit and mosquito mortality due to the insecticide is high, target other vector mosquito breeding habitats. More broadly, these experiments demonstrate that model systems provide an economical framework for testing different mosquito and mosquito-borne pathogen control strategies and can ultimately inform field interventions and enable better allocation of intervention resources.

West Nile virus (WNV) is currently active in Kenya as evidenced by the detection of antibodies in birds bled as part of an avian influenza surveillance program in 2009. Although WNV has been isolated from several mosquito species in Kenya, no studies have ever been conducted to determine which of these species are competent vectors of this virus. Therefore, we allowed Kenyan mosquitoes to feed on 2- or 3-d-old chickens that had been infected with a Lineage one strain of WNV 24–48 h earlier. These mosquitoes were tested ≈2 wk later to determine infection, dissemination, and transmission rates. All five species [Culex quinquefasciatus Say, Culex univittatus Theobald, Culex vansomereni Edwards, Mansonia africana (Theobald), and Mansonia uniformis (Theobald)] were susceptible to infection, but disseminated infections were detected only in the three Culex, and not the two Mansonia species. Culex mosquitoes with a disseminated infection readily transmitted virus by bite, but even when inoculated with WNV, the two Mansonia failed to transmit virus, indicating a salivary gland barrier. These studies indicate that the three Culex species may play a role in the transmission of WNV in Kenya.

The house fly (Musca domestica L.) may transmit Campylobacter to broiler flocks. We assessed the retention time of house flies for Campylobacter jejuni at five temperatures and three doses. Flies were inoculated individually at their proboscis with 1.6 × 10⁷ CFU (colony forming units) of C. jejuni and incubated at 15, 20, 25, 30, and 35°C. Furthermore, a dose experiment was conducted at 25°C where individual flies were inoculated in three series: 6.5 × 10⁶, 6.0 × 10⁴, and 8.2 × 10² C. jejuni CFU. Whole flies were tested for C. jejuni carriage at 0, 6, 12, 18, and 24 h by initial preenrichment in Bolton broth, which afterwards was streaked on modified mCCDA agar plates and incubated under micro-aerobic conditions. The results showed that the time C. jejuni remained in flies declined over time with ascending temperatures and when reducing the inoculation dose. All flies stayed Campylobacter positive 24 h postinoculation at 15°C whereas only one-third of the flies were positive at 20°C and few to none at 25, 30, and 35°C. When combinations of temperature and retention time were expressed as accumulated day-degrees, data could be adequately fitted using a generalized linear mixed model that included a linear effect of day-degrees and the difference between the lowest and the two highest doses. Based on model predictions of selected combinations of temperature and dose, the time for 50% and 1% of flies containing Campylobacter was calculated. It is suggested that house flies are mainly short distance carriers of C. jejuni.

Despite the importance of per-capita feeding rates for mosquito-borne transmission dynamics, the relationship between host aggregation and per-capita feeding rates remains poorly characterized. We conducted indoor experiments to investigate how Culex quinquefasciatus (Say) mosquitoes distribute their blood feeding on variably aggregated domestic chickens (Gallus gallus domesticus L.) (one chicken vs. a flock of seven to nine birds). Mosquitoes were always more likely to feed on the larger chicken group; yet, the single chicken tended to be fed on at a higher per-capita rate. When 10 chickens were available the feeding intensity was 4.5 times higher for the single chicken compared with the flock. We conclude that more highly aggregated hosts may experience lower exposure to mosquito bites than less aggregated hosts.

The population of Aedes albopictus (Skuse) (Diptera: Culicidae) was measured in four towns in northern Italy by means of Stegomyia indices (house index: percentage of houses with at least one active breeding site; container index: percentage of containers with larvae; and Breteau index: number of active breeding sites per premises), pupae per premises index (PPI), pupae per hectare index (PHI), and ovitrap monitoring. A detailed investigation on types and number of productive or potential larval habitats in the inspected premises was performed. Catch basins were the most productive breeding sites in both private and public areas. The influence of the premises maintenance and premises shading indices (PMI and PSI) on the population indices and the correlations between the latter and egg density were assessed. The highest PPI was found in badly maintained premises, most times combined with shady coverage. Stegomyia indices and PPI were well correlated with each other, but not with the PHI, which also included the pupae collected in public areas. We obtained a highly positive correlation (R = 0.86) between the PHI and weekly mean egg density estimated 7–14 d after inspection of the premises. The number of females per hectare, calculated using the Focks model, was correlated with the mean egg density of the week after sampling (R = 0.79), and we propose that ovitrap monitoring can be used to evaluate the efficiency of the control activities or to determine a mean egg density threshold for epidemic risk evaluation.

To determine whether a Sindbis virus (family Togaviridae, genus Alphavirus, SINV) infection in Aedes aegypti (L.) (Diptera: Culicidae) affected its response to the repellent DEET, we orally exposed Ae. aegypti to an artificial bloodmeal containing SINV or diluent and then allowed to feed on a 10% sucrose suspension containing 3% DEET. When tested seven or more days after the initial bloodmeal, although none of the diluent-exposed mosquitoes fed on the DEET-sucrose suspension, at least 60% of the SINV-exposed mosquitoes fed on the suspension. When legs from the SINV-exposed mosquitoes were tested to determine dissemination status, 89% of those that fed on the DEET-sucrose suspension contained virus. In contrast, only 34% of the nonfeeders had a disseminated infection. When offered a choice between sucrose with or without DEET, a significantly higher percentage of the SINV-exposed mosquitoes than the control mosquitoes fed on the sucrose containing 3% DEET. Together, these results indicate that mosquitoes with a disseminated SINV infection may be less responsive to DEET than uninfected mosquitoes. Therefore, repellent use may be less effective in deterring infected mosquitoes from biting than previously believed.

To have large numbers of replicates in forensic entomology research, it is often necessary to freeze carcasses and thaw them before use in experiments. Research on the effects of freezing on decomposition is minimal in the literature and the effect that it has on insect (e.g., blow fly, beetle) activity is virtually unknown. The objective of this study was to compare the differences in insect activity occurring on refrigerated only versus frozen-thawed pig carcasses, and to characterize the associated dipteran fauna. Seven pigs, frozen for 2 mo and then thawed before the experiment, were compared with seven pigs killed and stored in a walk in refrigerator ≈12 h before the start of the experiment. No significant differences in time to the appearance of adult flies, eggs, larvae, or the initiation and conclusion of larval migration were observed between refrigerated only and frozen-thawed pig carcasses. Beetles from the family Staphylinidae also did not show a significant difference in arrival times. Phormia regina (Meigen), Lucilia coeruleiviridis (Macquart), Cochliomyia macellaria (F.), Pollenia rudis (F.), and Hydrotaea leucostoma (F.) comprised the dipteran taxa present on the carcasses. Results suggest that freezing pigs before exposure in the field does not significantly alter blow fly life events or the appearance of staphylinid beetles.

There is accumulating evidence that criminals wrap dead bodies in an attempt to conceal evidence. To anticipate the forensic implications of this phenomenon, we examined whether flies that are naturally associated with cadavers exhibit a delay in attendance or differ in species composition and abundance patterns because of the presence of wrapping material. Wrapped and exposed carcasses of dead monkeys placed in an oil plantation in Kedah, Malaysia, were visited over 50 d. On daily visits to each of the six carcasses, visiting adult flies were sampled using hand nets. Flies of 12 families were encountered. Calliphoridae (Chrysomya rufifacies Macquart and C. megacephala (F.) was the most prevalent family, followed by Sphaeroceridae. Some families tended to be more abundant in WRCs (i.e., Calliphoridae, Muscidae, and Phoridae), whereas others (i.e., Piophilidae, Sepsidae, and Psychodidae) were more prevalent in exposed carcasses. Wrapping delayed the arrival of all fly species encountered, with delays varying from 1 to 13 d depending on species. Wrapping did not affect species composition of flies, but prolong the occurrence of some species. The results of the current study emphasize the need to take into consideration the presence of a wrap when estimating postmortem interval.

Three plant-based repellents—REPEL LEMON Eucalyptus Insect Repellent Lotion (active ingredient [AI] 30% oil of eucalyptus), Bite Blocker Xtreme Sportsman Organic Insect Repellent ([AI] 3% soybean oil, 6% geranium oil, and 8% castor oil), and Bite Blocker BioUD Insect Repellent ([AI] 7.75% 2-undecanone)—were evaluated against OFF! ([AI] 15% N,N-diethyl-m-toluamide or N,N-diethyl-3-methyl-benzamide, also called DEET) at a field site in Elkton, FL, to determine the mean protection time provided against Psorophora columbiae (Dyar & Knab). These products provided different protection times against biting Ps. columbiae. REPEL provided the longest protection time (330 min) followed by Bite Blocker Xtreme Sportsman (163 min), Bite Blocker BioUD (140 min), and OFF! (130 min). This study provides the first information about plant-based insect repellent protection times against Ps. columbiae.

In total, 22,846 (17,793 culicines and 5,053 Anopheles spp.) female mosquitoes were captured by a Mosquito Magnet trap at Daeseongdong, a small village adjacent to the military demarcation line (center of the demilitarized zone) in northern Gyeonggi Province, Republic of Korea (ROK). Culicine mosquitoes were identified to species, placed in pools of up to 30 mosquitoes each, and screened for flavivirus using a SYBR Green I-based real-time polymerase chain reaction. In total, 51/660 pools positive for flaviviruses and confirmed by DNA sequencing of the NS5 region, were positive for Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) (50 Culex tritaeniorhynchus Giles and one Culex bitaeniorhynchus Giles). The JEV maximum likelihood estimations (MLEs) (estimated number of viral RNA-positive mosquitoes per 1,000) for Cx. tritaeniorhynchus and Cx. bitaeniorhynchus were 9.7 and 0.9, respectively. This is the first report of a Cx. bitaeniorhynchus positive for JEV in the ROK. JEV is a local civilian and military health threat and a significant concern for nonimmune (unvaccinated) U.S. soldiers, civilians, and family members deployed to the ROK.

A total of 103 ticks, collected from canines, horses, donkeys, and snakes from Peru, were screened for the presence of Bartonella DNA by polymerase chain reaction analysis. Bartonella DNA was detected in two ticks using Bartonella 16S–23S intergenic spacer region primers and in an additional two ticks using Bartonella NADH dehydrogenase gamma subunit gene (nuoG) primers. Bartonella rochalimae Eremeeva et al., B. quintana Schmincke, and B. elizabethae Daly et al. DNA was detected in a Rhipicephalus sanguineus Latreille (Acari: Ixodidae) female tick removed from a dog and B. quintana DNA was present in a Dermacentor nitens Neumann (Acari: Ixodidae) pool of five larvae, one nymph, and one adult male tick collected from donkeys. This is the first study to report the detection of B. rochalimae, B. quintana, and B. elizabethae DNA in ticks from Peru. Further investigations must be performed to decipher the role ticks may play in the transmission of Bartonella species.