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A field strain of Aedes aegypti (L.) was collected from Puerto Rico in October 2008. Based on LD50 values by topical application, the Puerto Rico strain was 73-fold resistant to permethrin compared with a susceptible Orlando strain. In the presence of piperonyl butoxide, the resistance of Puerto Rico strain of Ae. aegypti was reduced to 15-fold, suggesting that cytochrome P450-mediated detoxification is involved in the resistance of the Puerto Rico strain to permethrin. To determine the cytochrome P450s that might play a role in the resistance to permethrin, the transcriptional levels of 164 cytochrome P450 genes in the Puerto Rico strain were compared with that in the Orlando strain. Of the 164 cytochrome P450s, 33 were significantly (P 0.05) up-regulated, including cytochrome P450s in families four, six, and nine. Multiple studies have investigated the functionality of family six and nine cytochrome P450s, therefore, we focused on the up-regulated family 4 cytochrome P450s. To determine whether up-regulation of the four cytochrome P450s had any functional role in permethrin resistance, transgenic Drosophila melanogaster Meigen lines overexpressing the four family 4 P450 genes were generated, and their ability to survive exposure to permethrin was evaluated., Publishe in the Journal of Medical Entomology, v51 n3 p605-615, May 2014. Prepared in collaboration with the Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, and USDA/ARS, Mosquito and Fly Research Unit, Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL.

With only 2 adulticide classes left to combat an increasingly resistant adult mosquito population and the pressing need for novel larvicides with new modes of action, a call has been initiated by the United States Department of Defense (DOD) and public health agencies to develop new classes of insecticides that are affordable and efficacious in killing mosquitoes, especially resistant ones. At the United States Department of Agriculture (USDA), several research units within the Agricultural Research Service (ARS) are actively involved in discovering and developing novel insecticides. These units include the Mosquito and Fly Research Unit (MFRU) at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) located at Gainesville, Florida; the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL) located at Beltsville, Maryland; and the Natural Products Utilization Research Unit (NPURU) located at Oxford, Mississippi. Funding for much of this research is provided by the DoD's Deployed War-fighter Protection (DWFP) Research Program, which seeks to develop insecticides with new modes of action for both military and civilian use. Discussed below are the insecticide research goals of these ARS units and the impressive results from their insecticide discovery programs.

An Aedes aegypti larval toxicity bioassay was performed on compounds representing many classes of natural compounds including polyacetylenes, phytosterols, flavonoids, sesquiterpenoids, and triterpenoids. Among these compounds, two eudesmanolides, alantolactone, and isoalantolactone showed larvicidal activities against Ae. aegypti and, therefore, were chosen for further structure?activity relationship study. In this study, structural modifications were performed on both alantolactone and isoalantolactone in an effort to understand the functional groups necessary for maintaining and/or increasing its activity, and to possibly lead to more effective insect-control agents. All parent compounds and synthetic modification reaction products were evaluated for their toxic activities against Ae. aegypti larvae and adults. Structure modifications included epoxidations, reductions, catalytic hydrogenations, and Michael additions to the a,b-unsaturated lactones. None of the synthetic isomers synthesized and screened against Ae. aegypti larvae were more active than isoalantolactone itself which had an LC50 value of 10.0 mg/ml. This was not the case for analogs of alantolactone for which many of the analogs had larvicidal activities ranging from 12.4 to 69.9 mg/ml. In general, activity trends observed from Ae. aegypti larval screening were not consistent with observations from adulticidal screening. The propylamine Michael addition analog of alantolactone was the most active adulticide synthesized with an LC50 value of 1.07 mg/mosquito. In addition, the crystal structures of both alantolactone and isoalantolactone were determined using CuKa radiation, which allowed their absolute configurations to be determined based on resonant scattering of the light atoms., Pub. in Chemistry and Biodiversity, v7, p1681-1697, 2010.

Aedes aegypti L. is the primary vector of dengue and yellow fever viruses, and use of aerosolized insecticides is one of the primary ways to control this medically important mosquito. However, few new insecticides have been developed for mosquito control in recent years. As a part of our effort to search for new insecticides to control mosquitoes, toxicities of 33 carboxamides were evaluated against female A. aegypti by topical application. This group included nine different categories of compounds, namely benzamides, phenyl-propenamides, propanamides, butanamides, butenamides, pentanamides, pentenamides, hexanamides, and hexenamides, that exhibited varying levels of toxicity against this mosquito species. The most toxic compound tested was hexahydro-1-(1-oxohexyl)-1H-azepine, with a 24-h LD50 value of 0.4 micrograms per mosquito, whereas the most toxic compound at the LD95 level was N-ethyl-2-methyl-N-phenyl-benzamide (1.82 micrograms per mosquito). The least toxic compound was N,N-bis (2-methylpropyl)-3-phenyl-2-propenamide, with LD50 and LD95 values of 15.66 and 72.07 micrograms per mosquito, respectively. Results from this initial study may prove useful in guiding further carboxamide modi cations for the development of potential new insecticides., Published in Journal of Medical Entomology, v47 n2 p172-178, Mar 2010.

Mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening. -Mangostin was tested for its larvicidal activity against third instar larvae of six mosquito species, and the median lethal concentration values range from 0.84 to 2.90 ppm. The residual larvicidal activity of -mangostin was examined under semifield conditions. The results indicated that -mangostin was photolytic with a half-life of 53 min in water under full sunlight exposure. The effect of -mangostin on activities of major detoxification enzymes such as P450, glutathione S-transferase, and esterase was investigated. The results showed that -mangostin significantly elevated activities of P450 and glutathione S-transferase in larvae, whereas it suppressed esterase activity. Toxicity of -mangostin against young rats was studied, and there was no detectable adverse effect at dosages as high as 80 mg/kg. This is the first multifaceted study of the biological activity of-mangostin in mosquitoes. The results suggest that-mangostin may be a lead compound for the development of a new organically based mosquito larvicide., Published in the Journal of Medical Entomology v47 n2 p249-257, 2010. Prepared in cooperation with the University of Wisconsin College of Agriculture

Ultra-low-volume (ULV) and thermal fog aerosol dispersals of pesticides have been used against mosquitoes and other insects for half a century. Although each spray technology has advantages and disadvantages, only 7 studies have been identified that directly compare their performance in the field. US military personnel currently operating in hot-arid environments are impacted by perpetual nuisance and disease vector insect problems, despite adulticide operations using modern pesticide-delivery equipment such as ULV. None of the identified comparative studies has looked at the relative feasibility and efficacy of ULV and thermal fog equipment against mosquitoes in hot-arid environments. In this study we examine the impact of ULV and thermal fog applications of malathion against caged sentinel mosquitoes in the field in a warm temperate area of Florida, followed by a similar test in a hot-dry desert area of southern California. Patterns of mortality throughout 150 m × 150 m grids of sentinel mosquitoes indicate greater efficacy from the thermal fog application in both environments under suboptimal ambient weather conditions. We discuss the implications of these findings for future military preventive medicine activities and encourage further investigations into the relative merits of the 2 technologies for force health protection.

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 deg 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., Published in the Journal of Medical Entomology, v46 n3 p490-495

The relative toxicities of 8 repellents (DMP, Rutgers 612, DEET, IR3535, Picardin, PMD AI3-35765, and AI3-37220) were evaluated by topical application against females of Aedes aegypti, Culex quinquefasciatus, Anopheles quadrimaculatus, and An. albimanus. Based on 24-h LD50 values, the most toxic repellent against all 4 mosquito species was AI3-37220, with values of 0.25, 0.20, 0.16, and 0.11 mg/mg for the listed 4 mosquito species, respectively. The least toxic of the 8 repellents tested was DMP, with LD50 values of 5.40, 4.72, 2.50, and 1.83 mg/mg, respectively. Based on the 24-h LD50 values, An. albimanus was the most susceptible species. The findings of the study reported herein provide a comprehensive examination of the toxicities of 4 currently used, 2 formerly used, and 2 experimental repellents against 4 mosquito species., Published in the Journal of the American Mosquito Control Association, v25 n2 p168-173, 2009.

Cytochrome b, coded by mitochondrial DNA, is one of the cytochromes involved in electron transport in the respiratory chain of mitochondria. Cytochrome b is a critical intermediate in a mitochondrial death pathway. To reveal whether cytochrome b of the mosquito Aedes aegypti (L.) (Diptera: Culicidae) (AeaCytB) is developmentally regulated, we used real-time quantitative polymerase chain reaction (qPCR) to examine AeaCytB gene expression levels in different developmental stages of Ae. aegypti. The qPCR showed that AeaCytB was expressed in each developmental stage, with peaks at first and second instars and was highly expressed in teneral male and female Ae. aegypti adults. Because mitochondrial genes exist as multiple copies, AeaCytB has much higher expression levels in all developmental stages in Ae. aegypti compared with nuclear genes. We also investigated the effect of abiotic environmental factors (e.g., high temperatures, ultraviolet radiation, and pesticide) on AeaCytB gene expression. Taken together, these results suggest that AeaCytB gene plays an important role in the development of Ae. aegypti and its response to environmental stress., Published in Journal of Medical Entomology, v46 n6 p1361-1369, Nov 2009.

Mosquitoes that transmit human diseases are of major importance to the international public health community. Pesticides remain a major component of integrated programs to control these medically important species. However, very few types of pesticides are currently registered for mosquito control. A high-throughput screening method using first-instar larvae of Aedes aegypti was created and evaluated in our laboratory to quickly screen large numbers of chemicals for activity against mosquitoes. LC50 values of a representative group of compounds were determined using this high-throughput screening method and compared with LD50 values determined by topical application against female adults of Ae. aegypti. Our results show that this high-throughput screening method is suitable for screening large numbers of candidate chemicals quickly to identify effective compounds., Published in the Journal of Medical Entomology v46 n2 p335-341, 2009.

The relative toxicities of 8 repellents (DMP, Rutgers 612, DEET, IR3535, Picardin, PMD, AI3-35765, and AI3-37220) were evaluated by topical application against females of Aedes aegypti, Culex quinquefasciatus, Anopheles quadrimaculatus, and An. albimanus. Based on 24-h LD50 values, the most toxic repellent against all 4 mosquito species was AI3-37220, with values of 0.25, 0.20, 0.16, and 0.11 µg/mg for the listed 4 mosquito species, respectively. The least toxic of the 8 repellents tested was DMP, with LD50 values of 5.40, 4.72, 2.50, and 1.83 µg/mg, respectively. Based on the 24-h LD50 values, An. albimanus was the most susceptible species. The findings of the study reported herein provide a comprehensive examination of the toxicities of 4 currently used, 2 formerly used, and 2 experimental repellents against 4 mosquito species.

Apoptosis (programmed cell death) is a tightly regulated physiological process. The inhibitors of apoptosis proteins (IAPs) are key regulators for apoptosis. An inhibitor of apoptosis protein gene IAP1 was recently cloned from Aedes aegypti (L.) (AaeIAP1, GenBank accession no. DQ993355); however, it is not clear whether AaeIAP1 is developmentally and environmentally regulated. In this study, we applied quantitative polymerase chain reaction (PCR) to investigate the expression levels of the AaeIAP1 transcript in different developmental stages and under different environmental conditions. Our results revealed that the expression of the AaeIAP1 transcript was detectable in all life stages of Ae. aegypti, with significantly higher levels in pupal and adult stages than in larval stages. Furthermore, when Ae. aegypti was exposed to all stressful environmental conditions (e.g., low and high temperatures, UV radiation, acetone, and permethrin insecticide treatment), the expression level of AaeIAP1 transcript was increased significantly. Our results suggest that AaeIAP1 might play an important role in both the physiological development of Ae. aegypti and stress-induced apoptosis., Pub. in Journal of Medical Entomology, v45 n6, p1071-1079, 2008.

To access the relative potency of pesticides to control adult mosquitoes, 19 pesticides with various modes of action were evaluated against Aedes aegypti, Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say. On the basis of 24-h LD50 values after topical application, the only pesticide that had higher activity than permethrin was fipronil, with LD50 values lower than permethrin for 107-, 4,849-, and 2-fold against Ae. aegypti, Cx. quinquefasciatus Say, and An. quadrimaculatus Say, respectively. Abamectin, imidacloprid, spinosad, diazinon, and carbaryl showed slightly lower activity than permethrin (<20-fold). However, bifenazate showed very low activity against the three mosquito species tested, with LD50 values higher than permethrin for 1000-fold. On the basis of 24-h LD50 values, Cx. quinquefasciatus was the least susceptible species to nine pesticides tested (DNOC, azocyclotin, chlorfenapyr, carbaryl, spinosad, imidaclorid, diazinon, abamectin, and permethrin), whereas Ae. aegypti was the least susceptible species to six pesticides tested (dicofol, amitraz, propargite, hydramethylnon, cyhexatin, and diafenthiuron), and An. quadrimaculatus was the least susceptible species to four pesticides tested (bifenazate, pyridaben, indoxacarb, and fipronil). Our results revealed that different species of mosquitoes had different susceptibility to pesticides, showing the need to select the most efficacious compounds for the least susceptible mosquito species to achieve successful mosquito control., Published in Journal of Medical Entomology, v45 n1 p82-87, Jan 2008.