Controlled release spatial repellent devices (CRDs) as novel tools against malaria transmission: a semi-field study in Macha, Zambia.

BACKGROUND: The emergence of mosquitoes that can avoid indoor-deployed interventions, such as treated bed nets and indoor residual spraying, threatens the mainstay of malaria control in Zambia. Furthermore, the requirement for high coverage of these tools poses operational challenges. Spatial repellents are being assessed to supplement these vector control tools, but limitations exist in the residual effect of the repellent and the need for external power or heat for diffusion of the volatiles. METHODS: A semi-field evaluation of a novel controlled release spatial repellent device (CRD) was conducted in Macha, Zambia. These devices emanate metofluthrin with no need for external power. Devices were deployed in huts within the semi-field system (SFS). Female Anopheles gambiae sensu stricto released within the SFS were trapped overnight by light traps and collected by aspiration the next morning inside and outside of huts to determine the extent of mosquito repellency and the impact on host-seeking and survival. Experiments studied the impact of number of devices as well as the presence of hut occupants. The study was complemented with numerical methods based on computational fluid dynamics to simulate spatial distribution of metofluthrin. RESULTS: Presence of CRDs was associated with significant reductions in indoor counts of mosquitoes, regardless of whether huts were occupied or not. Repellency ranged from 15 to 60% compared to huts with no devices. Reducing the number of devices from 16 to 4 had little impact on repellency. When huts were occupied, indoor mosquito host-seeking was higher in the presence of CRDs, whilst survival was significantly reduced. CONCLUSIONS: This study demonstrated that deployment of as few as four CRDs within a hut was associated with reduced indoor mosquito densities. As would be expected, presence of occupants within huts, resulted in greater indoor catches (both with and without devices). The increased indoor mosquito host-seeking and mortality in huts when devices were present may be explained by the excito-repellency activity of metofluthrin. These semi-field experiments provide preliminary data on the utility of CRD spatial repellents to reduce indoor densities of An. gambiae mosquitoes. Studies will further investigate the impact of CRDs on mosquito behaviour as well as epidemiological protective efficacy.

Evaluation of Pyriproxyfen Dissemination via Aedes albopictus From a Point-Source Larvicide Application in Northeast Florida.

The Asian tiger mosquito, Aedes albopictus , ranks among the most important vectors of dengue fever, Zika virus, and chikungunya virus. With no specific medications or vaccines available, vector control is the only way to combat these diseases. Autodissemination of the insect growth regulator pyriproxyfen (NyGuard®) from a point-source treatment was evaluated in field settings in northeast Florida. The objective of this study was to investigate the possibility of pyriproxyfen dissemination from a treatment site to nontreated oviposition sites via the skip oviposition behavior of Ae. albopictus. A spray application was made to a tire pile using a Stihl® SR 420 backpack sprayer. Autodissemination oviposition vases containing oak infusion water were positioned in groups of five at 25 to 400 m in 4 transects surrounding the tire pile. Two sets of 5 control vases containing oak infusion water were placed 1,500 m from the tire pile and oak infusion water samples were collected directly from the tire pile. Fifty milliliter samples were extracted from each vase weekly and preserved for pyriproxyfen residue analysis. All vases were analyzed at week 0 (4 h post-treatment), 1, 2, 4, and 6. Overall, there were no differences in pupal mortality between the control and autodissemination vases. The tire pile samples had significantly more mortality (P < 0.0001) out to 4 wk when compared to autodissemination and control vases.

Aedes albopictus (Diptera: Culicidae) Oviposition Preference as Influenced by Container Size and Buddleja davidii Plants.

Aedes albopictus (Skuse) is a container-breeding mosquito commonly found in residential areas of its range in the United States. Mosquitoes are known to utilize flowering plants for sugar acquisition. Limited information is known about the influences on oviposition site selection, outside of container size. Residential areas are often landscaped with a variety of flowering plants and are known to provide numerous sizes of potential larval developmental sites for container-breeding mosqutioes. Through screened enclosure and field studies, the oviposition preference of Ae. albopictus for containers of three selected sizes (473, 946 and 1,892 ml) and the influence of flowering butterfly bush (Buddleja davidii Franchett cultivar 'Guinevere') plants were examined. Our results document that significantly more eggs were oviposited in the largest containers. Additionally, significantly more eggs were oviposited in containers adjacent to flowering butterfly bushes than in those without a flowering butterfly bush. Finally, our results document that flowering butterfly bushes exerted greater influence over Ae. albopictus oviposition decisions than did container size. Our findings can be applied to several aspects of Ae. albopictus surveillance and control.

Development and Evaluation of an Attractive Self-Marking Ovitrap to Measure Dispersal and Determine Skip Oviposition in Aedes albopictus (Diptera: Culicidae) Field Populations.

Aedes albopictus (Skuse) is a container-breeding species with considerable public health importance. To date, Ae. albopictus oviposition behavior has been assessed in outdoor conditions, but only with laboratory-reared specimens. In outdoor large-cage and field studies, we used an attractive self-marking ovipositional device to assess Ae. albopictus skip oviposition behavior. In field studies, 37 wild Ae. albopictus that visited an attractive self-marking ovisite were subsequently captured at a sticky ovitrap within a 4-d period. Because the average Ae. albopictus gonotrophic period is 4.5-6 d, the wild-caught Ae. albopictus visited at least two oviposition sites within a single gonotrophic period. This provided field-based indirect evidence of skip oviposition. The mean distance traveled (MDT) during the 20-d evaluations ranged from 58 to 78 m. The maximum observed distance traveled was 149 m, which was the outer edge of our trapping ability. As populations of Ae. albopictus increased, the MDT during the 4- and 20-d post-marking period increased significantly. Additional observations of wild-marked and captured Aedes triseriatus (Say) are discussed.

The Effects of Larval Habitat Quality on Aedes albopictus Skip Oviposition.

Aedes albopictus is an invasive mosquito species that transmits human-disease-causing pathogens. It is a container-inhabiting species that oviposits in resource-limited habitats. To mitigate larval competition, Ae. albopictus females may choose to distribute eggs from a single gonotrophic cycle among multiple containers through skip oviposition. With the use of individual females released in indoor and outdoor caged trials, we evaluated the oviposition choices made by gravid Ae. albopictus offered larval habitats with different qualities. Our results demonstrate that Ae. albopictus performs skip oviposition and that the degree of egg distribution is related to the quality of the larval habitat. In a 4-choice arena, individual Ae. albopictus oviposited in fewer containers when presented with ovisites of high-quality larval habitat (uncrowded conditions) compared with oviposition in low-quality (crowded conditions) larval habitats. Additionally, the females selectively oviposited in high-quality habitats when offered both low- and high-quality habitats, but distributed eggs more evenly among multiple high-quality habitats. Our results have important implications for mosquito management plans that include the use of lethal ovitraps, as well as the role of this behavior in distribution of disease-causing pathogens.

Laboratory Studies of Aedes aegypti Attraction to Ketones, Sulfides, and Primary Chloroalkanes Tested Alone and in Combination with L-Lactic Acid.

The attraction of female Aedes aegypti to single compounds and binary compositions containing L-lactic acid and an additional saturated compound from a set of ketones, sulfides, and chloroalkanes was studied using a triple-cage dual-port olfactometer. These chemical classes were studied because of their structural relation to acetone, dimethyl disulfide, and dichloromethane, which have all been reported to synergize attraction to L-lactic acid. Human odors, carbon dioxide, and the binary mixture of L-lactic acid and CO2served as controls for comparison of attraction responses produced by the binary mixtures. All tested mixtures that contained chloroalkanes attracted mosquitoes at synergistic levels, as did L-lactic acid and CO2. Synergism was less frequent in mixtures of L-lactic acid with sulfides and ketones; in the case of ketones, synergistic attraction was observed only for L-lactic acid combined with acetone or butanone. Suppression or inhibition of attraction response was observed for combinations that contained ketones of C7-C12 molecular chain length (optimum in the C8-C10 range). This inhibition effect is similar to that observed previously for specific ranges of carboxylic acids, aldehydes, and alcohols.

Field Evaluation of a Novel Mos-Hole Trap and Naphtha Compared with BG Sentinel Trap and Mosquito Magnet X Trap to Collect Adult Mosquitoes.

The novel Mos-Hole trap™ with liquid attractant naphtha™ from Korea was compared with BG Sentinel™ trap and Mosquito Magnet X™ trap for field collection of adult mosquitoes in St. Johns County, northeastern Florida, from May to October 2013. The novel Mos-Hole trap baited with naphtha (liquid attractant) collected similar numbers of mosquitoes, compared with the number of mosquitoes caught by BG Sentinel traps baited with BG Lure™. Both Mos-Hole and BG Sentinel traps collected a significantly greater number of mosquitoes compared with the numbers collected by Mosquito Magnet X traps. In other field evaluations when switching lures, the Mos-Hole traps baited with BG Lure caught more mosquitoes than the BG Sentinel trap baited with liquid naphtha attractant. The results showed that the novel Mos-Hole trap has the potential to be used as an additional effective sampling tool for population surveillance and control of adult mosquitoes.

A Comparison of Carbon Dioxide Sources for Mosquito Capture in Centers for Disease Control and Prevention Light Traps on the Florida Gulf Coast (1).

Traditional sources of carbon dioxide (CO2), dry ice, and compressed gas, were tested against 3 combinations of food-grade reagents known to generate CO2using a compact, lightweight generator delivery system with Centers for Disease Control and Prevention light traps. Three 6 × 6 Latin square trials were completed near the Florida Gulf Coast in the Lower Suwannee Wildlife Refuge during the summer of 2013, collecting a total of 31,632 female mosquitoes. Treatments included dry ice, compressed CO2gas, a control trap (no CO2), citric acid + sodium bicarbonate, vinegar + sodium bicarbonate, and yeast + sugar. Decreasing order of trap collections (treatment mean number of mosquitoes per trap night ± standard error) were dry ice 773.5 (± 110.1) > compressed gas 440.7 (± 42.3) > citric acid + sodium bicarbonate 197.6 (± 30.4), yeast + sugar 153.6 (± 27.4) > vinegar + sodium bicarbonate 109.6 (± 16.2) > control 82.4 (± 14.0). A 2-way Kruskal-Wallis analysis by treatment, site, and treatment × site interaction identified significant differences between all treatments. Although dry ice and compressed CO2gas collected significantly more mosquitoes than other combinations (P < 0.05), use of citric acid and sodium bicarbonate or yeast and sugar greatly outperformed unbaited traps and offer a good alternative to dry ice and compressed gas in areas where these agents are not readily available or are difficult to obtain due to logistical constraints. An inexpensive, portable CO2generator for use with food-grade reagents is described.

Control of Aedes albopictus with attractive toxic sugar baits (ATSB) and potential impact on non-target organisms in St. Augustine, Florida.

The purpose of this study was to test the efficacy of bait stations and foliar applications containing attractive toxic sugar baits (ATSB) and eugenol to control Aedes albopictus. At the same time, the potential impact of these control methods was evaluated on non-target organisms. The study was conducted at five tire sites in St. Augustine, Florida. A. albopictus populations were significantly reduced with ATSB-eugenol applications applied directly to non-flowering vegetation and as bait stations compared with non-attractive sugar baits and control. The application of ATSB made to non-flowering vegetation resulted in more significant reductions of mosquito populations compared to the application of ATSB presented in a bait station. Over 5.5% of the non-targets were stained in the flowering vegetation application site. However, when the attractive sugar bait application was made to non-flowering vegetation or presented in bait stations, the impact on non-target insects was very low for all non-target orders as only 0.6% of the individual insects were stained with the dye from the sugar solutions, respectively. There were no significant differences between the staining of mosquitoes collected in flowering vegetation (206/1000) or non-flowering vegetation (242/1000) sites during the non-target evaluation. Our field studies support the use of eugenol as an active ingredient for controlling the dengue vector A. albopictus when used as an ATSB toxin and demonstrates potential use in sub-tropical and tropical environments for dengue control.

Field evaluation of four spatial repellent devices against Arkansas rice-land mosquitoes.

Four commercially available spatial repellent devices were tested in a rice-land habitat near Stuttgart, AR, after semi-field level assessments had been made at the Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, US Department of Agriculture in Gainesville, FL. OFF! Clip-On(metofluthrin), Mosquito Cognito (linalool), No-Pest Strip (dichlorvos), and ThermaCELL (d-cisltrans allethrin) were selected for this study from >20 candidate products. The units based on metofluthrin, linalool, or d-cisltrans allethrin significantly reduced captures of 1 or more of the mosquito species at surrogate human sites (unlit Centers for Disease Control and Prevention traps with CO2 and octenol). Among the mosquito species analyzed statistically (Anopheles quadrimaculatus, Culex erraticus, and Psorophora columbiae), there were significantly different responses (up to 84% reduction) to individual products, suggesting that combinations of certain spatial repellents might provide significantly greater protection.

Point Protection with Transfluthrin against Musca domestica L. in a Semi-Field Enclosure.

House flies are notoriously difficult to control, owing to their tendency to live in close relationships with humans and their livestock, and their rapid development of resistance to chemical controls. With this in mind, we explored an alternative chemical control, a spatial repellent to deter Musca domestica L. from points we wanted to protect (i.e., a baited trap). Our results demonstrated that the synthetic spatial repellent, transfluthrin, is effective in preventing M. domestica adults from entering protected traps for both a susceptible strain (CAR21) and a field-acquired permethrin-resistant strain (WHF; 24 h LD50 resistance ratio of 150), comprising 22% and 28% of the total number of flies collected, respectively. These results are promising and demonstrate that transfluthrin can be an effective spatial repellent to protect points of interest where needed.

Potential for mosquitoes (Diptera: Culicidae) from Florida to transmit Rift Valley fever virus.

We evaluated Aedes atlanticus Dyar and Knab, Aedes infirmatus Dyar and Knab, Aedes vexans (Meigen), Anopheles crucians Wiedemann, Coquillettidia perturbans (Walker), Culex nigripalpus Theobald, Mansonia dyari Belkin, Heinemann, and Page, and Psorophora ferox (Von Humboldt) from Florida to determine which of these species should be targeted for control should Rift Valley fever virus (RVFV) be detected in North America. Female mosquitoes that had fed on adult hamsters inoculated with RVFV were incubated for 7-21 d at 26 degrees C, then allowed to refeed on susceptible hamsters, and tested to determine infection, dissemination, and transmission rates. We also inoculated mosquitoes intrathoracically, held them for 7 d, and then allowed them to feed on a susceptible hamster to check for a salivary gland barrier. When exposed to hamsters with viremias > or = 10(7.6) plaque-forming units per milliliter of blood, at least some individuals in each of the species tested became infected; however, Cx. nigripalpus, An. crucians, and Ae. infirmatus were essentially incompetent vectors in the laboratory because of either a midgut escape or salivary gland barrier. Each of the other species should be considered as potential vectors and would need to be controlled if RVFV were introduced into an area where they were found. Additional studies need to be conducted with other geographic populations of these species and to determine how environmental factors affect transmission.

Comparison of Trap and Equine Attraction to Mosquitoes.

Mosquitoes are pests of horses, but mosquito trap efficacy data, especially the ability of traps to protect horses, are lacking. Studies were conducted to investigate the comparative attraction between traps and horses, increase trap attraction by adding horse odors to the airstream of a trap, determine the spatial distribution of adult mosquitoes, estimate the numbers of mosquitoes feeding on horses, determine the relative attraction of horses to mosquitoes, and estimate the range of mosquitoes' attraction between two horses. When a horse and a mosquito trap were placed 3.5 m apart, there was a significant reduction in mosquitoes entering the trap. Adding horse odors to the airstream of a trap produced equivocal results because the horse providing the odors influenced the trap catches. Mosquitoes were not evenly distributed across the study site, which emphasized the importance of trap placement. Vacuuming mosquitoes from the horses in different seasons demonstrated that 324 and 359 mosquitoes per hour were feeding during the two studies. Separate analysis of data from the two horses vacuumed simultaneously revealed that one horse attracted twice as many mosquitoes as the other. This caused the results of a study to determine the attraction range of two horses moved from 3.5 to 20.4 m apart to be inconclusive.

Semi-field evaluation of a novel controlled release device using transfluthrin as spatial repellent to prevent entry of mosquitoes into military tents.

Mosquitoes can impact military operational readiness by transmission of disease-causing pathogens or through secondary effects, e.g., annoyance and bites. The focus of this research was to determine if an array of novel controlled release passive devices (CRPD) utilizing the spatial repellent, transfluthrin (TF), as the active ingredient could prevent entry of mosquitoes into military tents for up to 4 weeks. The TF-charged CRPDs were spaced along six strands of monofilament and hung across the tent entrance. Efficacy was evaluated with caged Aedes aegypti to indicate knockdown/mortality effects, and four species of free-flying mosquitoes, Ae. aegypti, Aedes taeniorhynchus, Anopheles quadrimaculatus and Culex quinquefasciatus, to indicate repellent effects. Bioassay cages containing Ae. aegypti were hung vertically at 0.5, 1.0 and 1.5 â€‹m above ground level at designated locations inside of the tents. Knockdown/mortality counts were made every 15 min for the first hour, then at 2, 4 and 24 h post-exposure. Free fliers were recaptured in BG traps operated from 4 to 24 h post-exposure. Knockdown/mortality was gradual until 4 h post-exposure. This increased to near 100% by 24 h in the treated tent but was < 2% in the control tent. There was a significant reduction in the recapture rates of all free-flying species in the treated tent compared with the control tent. Results indicate that TF-charged CRPDs can significantly reduce the numbers of mosquitoes entering military tents and that the four species were affected similarly by the TF. The needs for additional research are discussed.

Elbow Injuries Among MLB Pitchers Increased During Covid-19 Disrupted Season, But Not Other Baseball Injuries.

Background: The 2020 Major League Baseball Season (MLB) demonstrated season disruptions due to the COVID-19 pandemic. Changes in training and seasonal time frames may be associated with higher rates of injury. Purpose: To use publicly available data to compare injury rates during the 2015-2019 seasons, COVID-19 shortened season (2020), and the 2021 season stratified by body region and position (pitchers versus position players). Study Design: A retrospective cohort study utilizing publicly available data. Methods: MLB players who competed in 1+ seasons between 2015-2021 were included and stratified by position (pitcher, position player). Incidence rate (IR), reported by 1000 x Athlete-Game Exposures (AGEs), was calculated for each season, and stratified by position and body region. Poisson regressions were performed for all injuries and stratified by position to determine association between season and injury incidence. Subgroup analyses were performed on the elbow, groin/hip/thigh, shoulder. Results: Four thousand, two hundred and seventy-four injuries and 796,502 AGEs across 15,152 players were documented. Overall IR was similar across seasons (2015-2019:5.39; 2020:5.85; 2021:5.04 per 1000 AGEs). IR remained high for the groin/hip/thigh for position players (2015-2019:1.7; 2020:2.0; 2021:1.7 per 1000 AGEs). There was no difference in injury rates between 2015-2019 and 2020 seasons [1.1 (0.9-1.2), p=0.310]. The 2020 season demonstrated a significant increase in elbow injuries [2.7 (1.8-4.0), p<0.001]; when stratified by position, this increase remained significant for pitchers [pitchers: 3.5 (2.1-5.9), p<0.001; position players: 1.8 (0.9-3.6), p=0.073]. No other differences were observed. Conclusion: The groin/hip/thigh demonstrated the highest IR in 2020 among position players across all season time frames, indicating that continued injury mitigation for this region is necessary. When stratified by body region, elbow injury rates among pitchers demonstrated 3.5 times the rate of injury in 2020 compared to previous seasons, impacting injury burden for the most vulnerable body region among pitchers. Level of Evidence: Level III.

Efficacy of three attractant blends tested in combination with carbon dioxide against natural populations of mosquitoes and biting flies at the Lower Suwannee Wildlife Refuge.

Synthetic blends of chemicals identified previously from human skin emanations were evaluated against mosquito and biting fly populations at the Lower Suwannee Wildlife Refuge near Cedar Key, FL. Mosquito Magnet-Experimental traps were baited with the Red (400 ml acetone: 10 ml 1-hexen-3-ol:10 ml 1-octen-3-ol), Blue (400 ml acetone: 1 g/liter lactic acid:20 ml glycolic acid), or Green blend (400 ml acetone:1.5 g/liter lactic acid:20 ml dimethyl disulfide) plus CO2 or with CO2 alone (control). A relative index of efficacy was determined by dividing each mean blend trap catch by the mean control trap catch. Five mosquitoes (Aedes infirmatus, Ae. taeniorhynchus, Ae. triseriatus, Anopheles crucians, and Culex nigripalpus), 2 ceratopogonid (Culicoides floridensis and C. furens), and 1 tabanid (Diachlorus ferrugatus) and phlebotomine (Lutzomyia shannoni) species were trapped. The Red blend + CO2 treatment significantly increased collections of Ae. taeniorhynchus (3.4x), An. crucians (2.8X), total mosquitoes (2.7x), C. furens (17.6x), and L. shannoni (10.8x) compared with control traps. Traps baited with either the Blue or Green blends generally captured fewer insects than traps baited with the other 2 treatments. However, traps baited with the Green blend caught 7 x as many C. furens as the control traps. Responses clearly varied according to species; therefore, "one size does not fit all" when it comes to attractant blends.

An innovative mosquito trap for testing attractants.

We describe a simple trap modification for testing volatile attractants to collect flying mosquitoes. The trap uses a standard Centers for Disease Control and Prevention trap modified for release of test chemicals. Test chemicals and other materials can be added and removed easily without spills or cross contamination. In preliminary studies using lactic acid and octenol, modified traps collected 40% more mosquitoes than controls (n = 164 and n = 117, respectively). Modifications cost less than $2.00 per trap.

Studies on the Volatiles Composition of Stored Sheep Wool, and Attractancy toward Aedes aegypti Mosquitoes.

To discover new natural materials for insect management, commercially available stored sheep wool was investigated for attractancy to female adult Aedes aegypti mosquitoes. The volatiles from sheep wool were collected by various techniques of headspace (HS) extractions and hydrodistillation. These extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detector (GC-FID) coupled with GC-MS. Fifty-two volatile compounds were detected, many of them known for their mosquito attractant activity. Seven compounds were not previously reported in sheep products. The volatile composition of the extracts varied significantly across collections, depending on the extraction techniques or types of fibers applied. Two types of bioassay were conducted to study attractancy of the sheep wool volatiles to mosquitoes: laboratory bioassays using glass tubes, and semi-field bioassays using large, screened outdoor cages. In bioassays with glass tubes, the sheep wool hydrodistillate and its main component, thialdine, did not show any significant attractant activity against female adult Ae. aegypti mosquitoes. Semi-field bioassays in two large screened outdoor cages, each equipped with a U.S. Centers for Disease Control (CDC) trap and the various bait setups with Vortex apparatus, revealed that vibrating wool improved mosquito catches compared to the setups without wool or with wool but not vibrating. Sheep wool, when vibrated, may release intensively volatile compounds, which could serve as olfactory cues, and play significant role in making the bait attractive to mosquitoes. Sheep wool is a readily available, affordable, and environment-friendly material. It should have the potential to be used as a mosquito management and surveillance component in dynamic bait setups.

Changes in host-seeking behavior of Puerto Rican Aedes aegypti after colonization.

The effects of colonization on host-seeking behavior of mosquitoes was examined by comparing attraction responses of newly colonized Aedes aegypti (L.) from field-collected eggs in Puerto Rico to that of the Gainesville (Florida) strain, originally from Orlando (Florida) and in colony since 1952. Females from the Orlando and the F0 through F10 generations of the Puerto Rico strain were evaluated using attractant odors in a triple-cage dual-port olfactometer. Two attractant sources were used: odors from the hand of a volunteer and a standard blend of L-lactic acid, acetone, and dimethyl disulfide. Convergence of the percentage of attraction responses occurred around the F4-F6 generations of the Puerto Rico strain. Both the Orlando and Puerto Rico strains exhibited similar responses for tests with the remaining F7-F10 generations. A temporal effect on mosquito responses was observed for both strains regardless of the attractant blend used in tests. This study indicates that Ae. aegypti host-seeking behavior changes significantly over the first four to six generations after introduction into the laboratory, whereas the field-collected strain increases in attraction response until it stabilizes at a new level.

Identification and transcription profiling of trypsin in Aedes taeniorhynchus (Diptera: Culicidae): developmental regulation, blood feeding, and permethrin exposure.

The cDNA of a trypsin gene from Aedes (Ochlerotatus) taeniorhynchus (Weidemann) was cloned and sequenced. The full-length mRNA sequence (890 bp) for trypsin from Ae. taeniorhynchus (AetTryp1) was obtained, which encodes an open reading frame of 765 bp (i.e., 255 amino acids). To detect whether AetTryp is developmentally regulated, a quantitative real-time polymerase chain reaction was used to examine AetTrypl mRNA expression levels in different developmental stages of Ae. taeniorhynchus. AetTryp1 was expressed at low levels in egg, larval, and pupal stages, but was differentially expressed in adult Ae. taeniorhynchus, with highest levels found in 5-d-old female adults when compared with teneral adults. In addition, AetTryp1 mRNA expression differed between sexes, with expression levels much lower in males. However, in both males and females, there was a significant increase in AetTryp1 transcription levels as age increased and peaked in 5-d-old adults. AetTrypl expressed in 5-d-old female Ae. taeniorhynchus significantly increased after 30 min postblood feeding compared with the control. The AetTryp1 mRNA expression in 5-d-old female Ae. taeniorhynchus was affected by different concentrations of permethrin.