Search

The repellency and toxicity of a CO2-derived cedarwood oil (CWO) was evaluated against actively questing unfed nymphs of four species of hard ticks: Amblyomma americanum (L.), Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus (Latreille). Using a vertical climb bioassay for repellency, nymphs of these species avoided a CWO-treated filter paper in proportional responses to treatment concentrations. At 60 min of exposure, I. scapularis nymphs were most sensitive with 50% repellency concentration (RC50) of 19.8 µg cm−2, compared with RC50 of 30.8, 83.8 and 89.6 µg cm−2 for R. sanguineus, D. variabilis and A. americanum, respectively. Bioassays determined the lethal concentration for 50% (LC50) and 90% (LC90) mortality of nymphs exposed to CWO in treated vials after 24- and 48-h exposure. After 24 h exposure, the LC50 values were 1.25, 3.45 and 1.42 µg cm−2 and LC90 values were 2.39, 7.59 and 4.14 µg cm−2 for D. variabilis, I. scapularis and R. sanguineus, respectively, but had minimal effect on A. americanum. After 48 h exposure, the LC50 values were 4.14, 0.78, 0.79 and 0.52 µg cm−2, and LC90 values were 8.06, 1.48, 1.54 and 1.22 µg cm−2 for A. americanum, D. variabilis, I. scapularis and R. sanguineus, respectively. The repellency of CWO on tick species decreased with time. The repellency and toxicity bioassays demonstrated concentration-dependent responses of tick nymphs to the oil, indicating the potential of the CO2-derived cedarwood oil be developed as an eco-friendly repellent and/or acaricide.

Stormwater drainage infrastructure creates abundant, nutrient-rich habitats in urban landscapes vulnerable to colonization by undesirable organisms, including vector mosquitoes and invasive aquatic macrophytes. The overall aim of our study was to identify consequences from cattails (Typha spp.) and their management to the seasonal abundance and adult fitness parameters of Culex pipiens and Cx. restuans developing in these surface stormwater habitats. We surveyed juvenile mosquitoes in 36 stormwater management structures (ditches and detention basins) with differing plant species composition and frequency of mowing. Effects of litter biomass, litter type (cattail, turfgrass) and exogenous enrichment with orthophosphate on oviposition site selection were evaluated in aquatic mesocosms. Individual laboratory experiments investigated effects of litter factors on juvenile development rate and survivorship, and adult body size and starvation resistance. We observed a greater abundance of both Cx. pipiens and Cx. restuans larvae in stormwater management structures with recently mowed vegetation. Additionally, we found a significant interaction between mowing and dominant plant type for Cx. pipiens, with the greatest number of larvae observed in mowed turfgrass habitats. For both Culex species, increasing litter biomass and the addition of orthophosphate enhanced oviposition rates in mesocosms, but the two species differed in their relative response to litter type. Similarly, we detected differences in the effects of litter type and biomass on Cx. pipiens and Cx. restuans development and adult fitness traits. Our results suggest asymmetrical effects from invasion and management of cattails in stormwater ditches and detention basins on potential risks posed by these two vector species.

Insecticide resistance has emerged as a persistent threat to the fight against vector-borne diseases. We compared the gut microbiota of permethrin-selected (PS) strain of Aedes aegypti relative to the parent (KW) strain from Key West, Florida. Bacterial richness but not diversity was significantly higher in PS strain compared to KW strain. The two mosquito strains also differed in their gut microbial composition. Cutibacterium spp., Corynebacterium spp., Citricoccus spp., Leucobacter spp., Acinetobacter spp., Dietzia spp., and Anaerococcus spp. were more abundant in PS strain than in KW strain. In contrast, Sphingomonas spp., Aquabacterium spp., Methylobacterium spp., Flavobacterium spp., Lactobacillus spp., unclassified Burkholderiaceae and unclassified Nostocaceae were more abundant in KW strain compared to PS strain. PS strain was enriched with propionate metabolizers, selenate reducers, and xylan, chitin, and chlorophenol degraders while KW strain was enriched with sulfur oxidizers, sulfur metabolizers, sulfate reducers and naphthalene and aromatic hydrocarbons degraders. These findings demonstrate an association between the gut microbiota and insecticide resistance in an important vector species and sets the foundation for future studies to investigate the contribution of gut microbiota to evolution of insecticide resistance in disease vectors.

Background The guts of blood-sucking insects host a community of bacteria that can shift dramatically in response to biotic and abiotic factors. Identifying the key factors structuring these microbial communities has important ecological and epidemiological implications. Methods We used the yellow fever mosquito, Aedes aegypti, to investigate the impact of mixed blood meals on gut microbiota of vector mosquitoes. Adult females were experimentally fed on sugar or blood from chicken, rabbit or a mixture of chicken and rabbit blood, and their gut microbiota were characterized using 16S rRNA gene amplification and MiSeq sequencing. Results The gut bacterial communities of mosquitoes fed on the three blood meal treatments clustered separately, suggesting that host species identity and mixed blood-feeding are key determinants of gut bacterial community composition in mosquitoes. Mixed blood meal had a synergistic effect on both operational taxonomic unit (OTU) richness and the Shannon diversity index, suggesting that mixed blood-feeding can offset the nutritional deficit of blood meals from certain host species. The microbial communities observed in this study were distinct from those identified from similarly fed Ae. aegypti from our previous study. Conclusions These findings demonstrate that vector host-feeding preferences can influence gut microbial composition and diversity, which could potentially impact pathogen acquisition and transmission by the vector. The results also demonstrate that different microenvironmental conditions within the laboratory may play an important role in structuring the microbial communities of independently reared mosquito colonies. Graphical Abstract

The use essential oils as ecofriendly tools for pest and vector management has become one of the mainstreams for biopesticide research. Here, Commiphora erythraea (opoponax) essential oil and its fractions were profiled for their chemical composition and larvicidal activity against Culex restuans, Cx. pipiens, and Aedes aegypti mosquitoes. The effect of C. erythraea essential oil on expression of three cytochrome P450 (CYP450s) and three Glutathione-S-transferases (GSTs) genes and the use of bio-based amylose-N-1-hexadecylammonium chloride inclusion complex (Hex-Am) and amylose-sodium palmitate inclusion complex (Na-Palm) as emulsifiers for C. erythraea essential oil were also investigated. The most abundant chemical constituents were curzerene (32.6%) in fraction 1, spathulenol (62.5%) in fraction 2, and bicyclosesquiphellandrene in the whole essential oil (33.9%) and factions 3 (30.1%) and 4 (23.8%). LC50 values for the whole essential oil were 19.05 ppm for Cx. restuans, 22.61 ppm for Cx. pipiens and 29.83 ppm for Ae. aegypti and differed significantly from each other. None of the four C. erythraea essential oil fractions were active against mosquito larvae. Two CYP450 genes (CYP6M11 and CYP6N12) and one GST gene (GST-2) were significantly upregulated in Ae. aegypti larvae exposed to C. erythraea essential oil suggesting their potential involvement in pathways of metabolic activation and detoxification of C. erythraea essential oil. Essential oil emulsions produced with Hex-Am were more toxic than the whole essential oil while those produced with Na-Palm had similar toxicity as the whole essential oil. These findings demonstrate that C. erythraea essential oil is a promising source of mosquito larvicide and that its larvicidal activity is likely due to synergistic interactions among the major and minor constituents. The findings also demonstrate that the use of Hex-Am as an emulsifier can enhance the insecticidal properties of C. erythraea essential oil.

A general method to spin-label a fatty acid was demonstrated as well as an assay of the effectiveness of methyl 10-undecenoate and the spin-labeled version, against the larvae of Aedes aegypti. The LC

The repellency and toxicity of a CO

Background Some plant essential oils have insecticidal properties against mosquitoes and can be harnessed as ecofriendly tools for mosquito control. We conducted bioassays to determine the toxicity of Italian honeysuckle (Lonicera caprifolium) essential oil and its fractions against larvae of the yellow fever mosquito, Aedes aegypti. Results Sixteen constituents were identified in honeysuckle essential oil compared to 15, 15, 15, and 11 constituents in fractions A, B, C, and E, respectively. The chemical constituents for fraction D were not determined due to lack of enough fraction sample. The two major constituents identified were patchouli alcohol (29.3%) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (20.6%) in whole essential oil, alpha-bulnesene (27.6%) and 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (23.2%) in fraction A, unknown chemical (47.3%) and diethyl phthalate (19.5%) in fraction B, unknown chemical (38.3%) and diethyl phthalate (23.2%) in fraction C, and patchouli alcohol (58.7%) and diethyl phthalate (20.5%) in fraction E. The LC50 for whole essential oil was 34.4 ppm and significantly higher than 20.6, 19.7, 18.6, and 17.7 ppm for fractions B, C, D, and E, respectively. In contrast, fraction A was inactive. At 50 ppm, all individual constituents tested were less toxic than the whole essential oil with exception of patchouli alcohol, which caused 100% mortality. Conclusion These findings suggest that patchouli alcohol is one of the chemical constituents responsible for bioactivity of honeysuckle essential oil and some of its fractions. The findings also demonstrate that honey suckle essential oil and its fractions can be exploited as a source of ecofriendly larvicides for mosquito control. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Background The rapid and widespread evolution of insecticide resistance has emerged as one of the major challenges facing malaria control programs in sub-Saharan Africa. Understanding the insecticide resistance status of mosquito populations and the underlying mechanisms of insecticide resistance can inform the development of effective and site-specific strategies for resistance prevention and management. The aim of this study was to investigate the insecticide resistance status of Anopheles gambiae (s.l.) mosquitoes from coastal Kenya. Methods Anopheles gambiae (s.l.) larvae sampled from eight study sites were reared to adulthood in the insectary, and 3- to 5-day-old non-blood-fed females were tested for susceptibility to permethrin, deltamethrin, dichlorodiphenyltrichloroethane (DDT), fenitrothion and bendiocarb using the standard World Health Organization protocol. PCR amplification of rDNA intergenic spacers was used to identify sibling species of the An. gambiae complex. The An. gambiae (s.l.) females were further genotyped for the presence of the L1014S and L1014F knockdown resistance (kdr) mutations by real-time PCR. Results Anopheles arabiensis was the dominant species, accounting for 95.2% of the total collection, followed by An. gambiae (s.s.), accounting for 4.8%. Anopheles gambiae (s.l.) mosquitoes were resistant to deltamethrin, permethrin and fenitrothion but not to bendiocarb and DDT. The L1014S kdr point mutation was detected only in An. gambiae (s.s.), at a low allelic frequency of 3.33%, and the 1014F kdr mutation was not detected in either An. gambiae (s.s.) or An. arabiensis. Conclusion The findings of this study demonstrate phenotypic resistance to pyrethroids and organophosphates and a low level of the L1014S kdr point mutation that may partly be responsible for resistance to pyrethroids. This knowledge may inform the development of insecticide resistance management strategies along the Kenyan Coast. Graphical Abstract

Pesticides commonly contaminate the aquatic environments inhabited by mosquito juveniles. However, their role in shaping the mosquito microbiota is not well understood. We hypothesized that environmentally relevant concentrations of atrazine, permethrin and malathion will mediate a shift in the mosquito gut bacterial community structure due to their toxic effect on the aquatic bacterial communities, and reduce mosquito gut bacterial diversity by enriching pesticide-degrading bacterial communities over susceptible taxa. Illumina MiSeq sequencing of the V3-V4 hypervariable regions of the 16 S rRNA gene was used to characterize the microbial communities of larval and adult stages of the two mosquito species and the water samples from microcosms treated with each of the pesticides, separately. Bacterial community composition differed by sample type (larval stage vs. adult stage) and water sampling date (day 3 vs. day 7), but not by pesticide treatment. In larval stages, bacterial OTU richness was highest in samples exposed to malathion, intermediate in permethrin, and lowest in controls. Bacterial richness was significantly higher in larval stages compared to adult stages for all treatments. This study provides a primer for future studies evaluating mosquito microbial responses to exposures to chemical pesticides and the possible implications for mosquito ecology.

Synthetic pesticides are the cornerstone of vector-borne disease control, but alternatives are urgently needed to tackle the growing problem of insecticide resistance and concerns over environmental safety. Leptospermum scoparium J.R. Forst and G. Forst (manuka) essential oil and its four fractions were analyzed for chemical composition and toxicity against Aedes aegypti larvae. The use of bio-based amylose-N-1-hexadecylammonium chloride inclusion complexes (Hex-Am) as an emulsifier for L. scoparium essential oil was also investigated. Fraction 1 was inactive, fractions 2 (LC50 = 12.24 ppm) and 3 (LC50 = 20.58 ppm) were more toxic than the whole essential oil (LC50 = 47.97 ppm), and fraction 4 (LC50 = 35.87 ppm) had similar toxicity as the whole essential oil. Twenty-one chemical constituents were detected in L. scoparium essential oil compared to 16, 5, 19 and 25 chemical constituents in fractions, 1, 2, 3 and 4 respectively. The two most dominant chemical constituents were calamenene (17.78%) and leptospermone (11.86%) for L. scoparium essential oil, calamenene (37.73%) and ledene (10.37%) for fraction 1, leptospermone (56.6%) and isoleptospermone (19.73) for fraction 2, cubenol (24.30%) and caryophyllene oxide (12.38%) for fraction 3, and γ-gurjunene (21.62%) and isoleptospermone (7.88%) for fraction 4. Alpha-pinene, ledene, and aromandendrene were 2-7 times less toxic than the whole essential suggesting that the toxicity of L. scoparium essential oil was either due to other chemical constituents that were not tested or due synergist interactions among chemical constituents. Leptospermum scoparium essential oil-Hex-Am emulsion (LC50 = 29.62) was more toxic than the whole essential oil. These findings suggest that L. scoparium essential oil is a promising source of mosquito larvicide and that Hex-Am is an excellent emulsifier for L. scoparium essential oil for use as a larvicide.

Background Understanding the major predictors of disease vectors such as mosquitoes can guide the development of effective and timely strategies for mitigating vector-borne disease outbreaks. This study examined the influence of selected environmental, weather and sociodemographic factors on the spatial and temporal distribution of the southern house mosquito Culex quinquefasciatus Say in New Orleans, Louisiana, USA. Methods Adult mosquitoes were collected over a 4-year period (2006, 2008, 2009 and 2010) using CDC gravid traps. Socio-demographic predictors were obtained from the United States Census Bureau, 2005–2009 American Community Survey and the City of New Orleans Department of Code Enforcement. Linear mixed effects models and ERDAS image processing software were used for statistical analysis and image processing. Results Only two of the 22 predictors examined were significant predictors of Cx. quinquefasciatus abundance. Mean temperature during the week of mosquito collection was positively associated with Cx. quinquefasciatus abundance while developed high intensity areas were negatively associated with Cx. quinquefasciatus abundance. Conclusion The findings of this study illustrate the power and utility of integrating biophysical and sociodemographic data using GIS analysis to identify the biophysical and sociodemographic processes that increase the risk of vector mosquito abundance. This knowledge can inform development of accurate predictive models that ensure timely implementation of mosquito control interventions.

Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait locus was cloned from an inbred with reported resistance to Fusarium proliferatum and Fusarium verticillioides ear rot. Transgenic expression of the gene in maize callus reduced colonization by these two Fusarium species and also Fusarium graminearum relative to a β-glucuronidase (GUS) transformant control. Some transformants were also more insect resistant. The more fungal resistant transformant lines produced higher levels of headspace ethanol which were significantly associated with antifungal activity, especially for F. verticillioides. Maize pyruvate decarboxylase appears to have a moiety capable of interacting with the geranyl geranyl transferase, suggesting ethanol production is enhanced due to more efficient transfer of pyruvate through the mitochondrial membrane. Other undetermined mechanisms may also be enhancing resistance of the transformants to the Fusarium fungus, however. This is the first report of the involvement of a geranyl geranyl transferase-like sequence in fungal resistance in plants, and represents a novel mechanism for producing higher yielding and better quality maize.

The potential for gut microbiota to impede or enhance pathogen transmission is well-documented but the factors that shape this microbiota in mosquito vectors are poorly understood. We characterized and compared the gut microbiota of adult females of Culex restuans (Theobald; Diptera: Culicidae) from different parents. Cx. restuans larvae from nine field-collected egg rafts were reared on a common diet and gut microbiota of newly emerged adult females characterized by MiSeq sequencing of the V4 hypervariable region of the 16S rRNA gene. Bacterial diversity and evenness in individuals from one egg raft were significantly lower compared to those of three of the other eight egg rafts. The gut microbiota of adult females reared from seven of the nine egg rafts clustered together suggesting that individuals from most egg rafts had similar profiles of gut microbiota. These findings suggest that the microbiota of adult females from the same parents do not differ appreciably from the microbiota of adult females from different parents. However, additional studies using mosquitoes separated by geographic distances greater than those studied here and estimating the genetic distances between populations from different egg rafts are needed to provide further insights into the influence of host genetics on gut microbiota. Also worthwhile are studies evaluating how individuals from different egg rafts and harboring different gut microbiota compare in relation to vector competence for different pathogens.

Essential oils are potential alternatives to synthetic insecticides because they have low mammalian toxicity, degrade rapidly in the environment, and possess complex mixtures of bioactive constituents with multi-modal activity against the target insect populations. Twenty-one essential oils were initially screened for their toxicity against Aedes aegypti (L.) larvae and three out of the seven most toxic essential oils (Manuka, oregano, and clove bud essential oils) were examined for their chemical composition and combined toxicity against Ae. aegypti larvae. Manuka essential oil interacted synergistically with oregano essential oil and antagonistically with clove bud essential oil. GC-MS analysis revealed the presence of 21 components in Manuka essential oil and three components each in oregano and clove bud essential oils. Eugenol (84.9%) and eugenol acetate (9.6%) were the principal constituents in clove bud essential oil while carvacrol (75.8%) and m-isopropyltoluene (15.5%) were the major constituents in oregano essential oil. The major constituents in Manuka essential oil were calamenene (20%) and 3-dodecyl-furandione (11.4%). Manuka essential oil interacted synergistically with eugenol acetate and antagonistically with eugenol, suggesting that eugenol was a major contributor to the antagonistic interaction between Manuka and clove bud essential oils. In addition, Manuka interacted synergistically with carvacrol suggesting its contribution to the synergistic interaction between Manuka and oregano essential oils. These findings provide novel insights that can be used to develop new and safer alternatives to synthetic insecticides.

We examined the growth characteristics of Tolypocladium cylindrosporum IBT 41712 and its potential to infect eggs of Aedes aegypti and Ae. albopictus at a low temperature (15°C). When grown on Sabouraud dextrose agar supplemented with yeast extract, the IBT 41712 formed white colonies turning to a slightly darker, off-white color when mature. The mycelia bore swollen conidiophores producing smooth-walled, oblong to cylindrical conidia with varying sizes, ranging from 1.5 to 3.5 μm long. To determine the optimum temperature for the fungus, we cultured the fungus at eight temperatures (4°C, 12°C, 15°C, 21°C, 28°C, 33°C, 37°C, and 40°C) and measured the diametric growth. The optimum temperature for growth was 28°C since it had the highest diametric growth rate (2.1 ± 0.05 mm/day) and the fastest sporulation period (within 8-10 days of incubation). There was no fungal growth at the 3 highest temperatures (33°C, 37°C, and 40°C) but plates incubated at 33°C, when shifted to optimal temperature (28°C), showed visible growth indicating that following incubation at 33°C, the fungus remained viable. The IBT 41712 successfully infected mosquito eggs at 15°C. Fungal treatment induced egg hatch on moist seed-germination paper and this effect was more pronounced in Ae. aegypti compared to Ae. albopictus. When treated eggs were immersed in dH2O 21 days posttreatment, larval hatch of both Ae. aegypti (control = 91%, 1 × 107 conidia/ml, fungal treatment = 0%) and Ae. albopictus (control = 85%, fungal treatment = 28%) was significantly lower in fungal treatment compared to the controls. The ability of the strain to grow in a wide temperature range, and effectively infect mosquito eggs and induce egg hatch at a low temperature warrants further investigation for its potential as a mosquito control agent targeting eggs that overwinter or undergo long diapause.

Summary Surveillance for West Nile virus (WNV) and other mosquito-borne pathogens involves costly and time-consuming collection and testing of mosquito samples. One difficulty faced by public health personnel is how to interpret mosquito data relative to human risk, thus leading to a failure to fully exploit the information from mosquito testing. The objective of our study was to use the information gained from historic West Nile virus mosquito testing to determine human risk relative to mosquito infection and to assess the usefulness of our mosquito infection forecasting models to give advance warning. We compared weekly mosquito infection rates from 2004 to 2013 to WNV case numbers in Illinois. We then developed a weather-based forecasting model to estimate the WNV mosquito infection rate one to 3 weeks ahead of mosquito testing both statewide and for nine regions of Illinois. We further evaluated human illness risk relative to both the measured and the model-estimated infection rates to provide guidelines for public health messages. We determined that across 10 years, over half of human WNV cases occurred following the 29 (of 210) weeks with the highest mosquito infection rates. The values forecasted by the models can identify those time periods, but model results and data availability varied by region with much stronger results obtained from regions with more mosquito data. The differences among the regions may be related to the amount of surveillance or may be due to diverse landscape characteristics across Illinois. We set the stage for better use of all surveillance options available for WNV and described an approach to modelling that can be expanded to other mosquito-borne illnesses.

Invasive species rank second only to habitat destruction as a threat to native biodiversity. One consequence of biological invasions is altered risk of exposure to infectious diseases in human and animal populations. The distribution and prevalence of mosquito-borne diseases depend on the complex interactions between the vector, the pathogen, and the human or wildlife reservoir host. These interactions are highly susceptible to disturbance by invasive species, including terrestrial plants. We conducted a 2-year field experiment using a Before-After/Control-Impact design to examine how removal of invasive Amur honeysuckle (Lonicera maackii) in a forest fragment embedded within a residential neighborhood affects the abundance of mosquitoes, including two of the most important vectors of West Nile virus, Culex pipiens and Cx. restuans. We also assessed any potential changes in avian communities and local microclimate associated with Amur honeysuckle removal. We found that (1) removal of Amur honeysuckle reduces the abundance of both vector and non-vector mosquito species that commonly feed on human hosts, (2) the abundance and composition of avian hosts is altered by honeysuckle removal, and (3) areas invaded with honeysuckle support local microclimates that are favorable to mosquito survival. Collectively, our investigations demonstrate the role of a highly invasive understory shrub in determining the abundance and distribution of mosquitoes and suggest potential mechanisms underlying this pattern. Our results also give rise to additional questions regarding the general impact of invasive plants on vector-borne diseases and the spatial scale at which removal of invasive plants may be utilized to effect disease control.

Although the insecticidal properties of some plant essential oils are well-documented, their use in integrated pest and vector management is complicated by their high volatility, low thermal stability, high sensitivity to oxidation, and low solubility in water. We investigated the use of bio-based N-1-hexadecylammonium chloride and sodium palmitate amylose inclusion complexes as emulsifiers for two essential oils, garlic and asafoetida, known to be highly toxic to mosquito larvae. Four emulsions of each essential oil based on amylose hexadecylammonium chloride and amylose sodium palmitate inclusion complexes were evaluated for their toxicity against Aedes aegypti L. larvae relative to bulk essential oils. All emulsions were significantly more toxic than the bulk essential oil with the lethal dosage ratios ranging from 1.09&ndash, 1.30 relative to bulk essential oil. Droplet numbers ranged from 1.11 &times, 109 to 9.55 &times, 109 per mL and did not change significantly after a 6-month storage period. These findings demonstrated that amylose inclusion complexes enhanced the toxicity of essential oils and could be used to develop new essential oil based larvicides for use in integrated vector management.

The growing awareness that microbial symbionts residing in mosquito midguts can interrupt transmission of vector-borne diseases has stimulated interest in understanding their potential role in mosquito biology. Fluorescent proteins are powerful molecular markers that provide for detailed analysis of the function and behavior of specific midgut bacterial isolates without disturbing the normal gut microbiota. The aim of this study was to label bacterial isolates from the midgut of Ochlerotatus triseriatus, the primary vector of La Crosse virus, with green, yellow, and red fluorescent proteins (GFP, YFP, RFP) via electroporation. We also assessed the stability of GFP-, YFP-, and RFP-bearing plasmids and their effect on bacterial growth. Seven of eleven bacterial species could not be labeled despite several attempts. Labeling of Escherichia coli and Enterobacter cloacae was successfully achieved with all three fluorescent proteins. In contrast, labeling of Aerococcus viridans was achieved with GFP only and labeling of Aeromonas hydrophila was achieved with GFP and YFP only. The stability of GFP plasmid varied among bacterial species with A. hydrophila followed by E. cloacae having the most stable GFP label. In contrast, YFP and RFP plasmids were very stable in all bacterial species possessing these labels. GFP plasmid reduced the growth of labeled strains relative to wild type but this effect was not evident in YFP and RFP plasmids. These findings suggest that some mosquito midgut bacterial isolates can effectively be labeled with GFP, YFP and RFP plasmids allowing non-destructive studies on their functions within the vector.

The use of microbial biocontrol agents as biopesticides has become an attractive insect pest management choice due to concerns regarding effects of chemical pesticide residues on human and animal health. In the biopesticide approach, an endemic microbial pathogen of an insect pest is identified that is capable of rapidly infecting and killing the target pest when the focal area is inundated. Initial screenings focus on finding aggressive pathogens of the target insect pest. The host range of these pathogens is then evaluated to ensure their safety. Formulations are subsequently developed to ensure effective application in the field. Once developed, the microbial biocontrol agent is used in a manner similar to a chemical pesticide. The mode of action, however, is such that the infective propagules produce multiple infections in the insect pest resulting in death. Because the biocontrol agent is restricted in its host range, only microbial propagules which contact and infect susceptible pests survive. Long-term persistence in the environment normally does not occur, so typically the inundation approach to microbial biocontrol does not produce multi-year control of the pest. Thus, seasonal application of the biocontrol agent is required, which provides the commercial incentive for development.

Emergence may involve invasive mosquitoes other than Asian tiger mosquitoes, climate change, and changes in wildlife densities., La Crosse encephalitis is a viral disease that has emerged in new locations across the Appalachian region of the United States. Conventional wisdom suggests that ongoing emergence of La Crosse virus (LACV) could stem from the invasive Asian tiger (Aedes albopictus) mosquito. Efforts to prove this, however, are complicated by the numerous transmission routes and species interactions involved in LACV dynamics. To analyze LACV transmission by Asian tiger mosquitoes, we constructed epidemiologic models. These models accurately predict empirical infection rates. They do not, however, support the hypothesis that Asian tiger mosquitoes are responsible for the recent emergence of LACV at new foci. Consequently, we conclude that other factors, including different invasive mosquitoes, changes in climate variables, or changes in wildlife densities, should be considered as alternative explanations for recent increases in La Crosse encephalitis.

Invasive mosquito species can increase the transmission risk of native mosquito-borne diseases by acting as novel vectors. In this study, we examined the susceptibility of three exotic invasive mosquito species Aedes aegypti (L.), Ae. albopictus (Skuse), and Ochlerotatus japonicus (Theobald) to La Crosse virus (LACV) relative to the native primary vector Ochlerotatus triseriatus (Say). Adult females of the four mosquito species were orally challenged with LACV; incubated for 3, 5, 7, 9, or 11 d; and their midgut infection rates, dissemination rates, and effective vector competence were determined. Overall, Oc. japonicus (2.92) had the highest effective vector competence values, followed by Ae. albopictus (1.55), Ae. aegypti (0.88), and Oc. triseriatus (0.64) . In addition, we assessed the relationship between mosquito size and LACV susceptibility for field-collected Oc. triseriatus and Oc. japonicus . We hypothesized that smaller adults would be more susceptible to LACV; however, our results did not support this hypothesis. Infected Oc. triseriatus tended to be larger than exposed but uninfected females, while infected and uninfected Oc. japonicus were similarly sized. These findings suggest that Oc. japonicus , Ae. albopictus , and Ae. aegypti have significant potential to transmit LACV and more research is needed to uncover their potential role in LACV epidemiology.

Artificial aquatic habitats are ubiquitous in anthropogenic landscapes and highly susceptible to colonization by invasive plant species. Recent research into the ecology of infectious diseases indicates that the establishment of invasive plant species can trigger ecological cascades which alter the transmission dynamics of vector-borne pathogens that imperil human health. Here, we examined whether the presence or management of two invasive, emergent plants, cattails (Typha spp.) and phragmites (Phragmites australis), in stormwater dry detention basins (DDBs) alter the local distribution of vectors, avian hosts, or West Nile virus (WNV) transmission risk in an urban residential setting. Mosquitoes and birds were surveyed at 14 DDBs and paired adjacent residential sites. During the study period, emergent vegetation was mowed by site managers in three DDBs. In the absence of vegetation management, the overall abundance and species composition of both adult vectors and avian hosts differed between residential and DDB habitats; however, WNV entomological risk indices were equivalent. Communal bird roosts composed primarily of three species, European Starlings (Sturnus vulgaris), Red-winged Blackbirds (Agelaius phoeniceus), and Common Grackles (Quiscalus quiscula), representing a broad range of WNV reservoir competence, were observed at half (three out of six) of the DDBs containing unmanaged stands of phragmites; however, their presence was associated with a lower seasonal increase in vector infection rate. Conversely, mowing of emergent vegetation resulted in a significant and sustained increase in the abundance of WNV-infected vectors in DDBs and the increase in risk extended to adjacent residential sites. These findings indicate that management of invasive plants in DDBs during the growing season can increase, while presence of communal bird roosts can decrease, WNV transmission risk.

Gut microbial communities of mosquitoes can influence vector susceptibility to pathogens, yet the factors that govern their composition remain poorly understood. We investigated the impact of host blood-meal source on gut microbiota of Aedes aegypti L. Adult mosquitoes were fed on human, rabbit or chicken blood and their gut microbiota compared to those of sugar-fed and newly emerged adults. Microbial diversity was significantly reduced in blood-fed and sugar-fed mosquitoes but was restored to the levels of newly emerged adults at 7-days post-blood meal. Microbial composition was strongly influenced by host blood-meal source. Leucobacter spp., Chryseobacterium spp., Elizabethkingia spp. and Serratia spp. were characteristic of newly emerged adults and adults fed on chicken, rabbit and human blood, respectively. Sugar-fed mosquitoes had higher abundance of Pseudomonas spp. and unclassified Acetobacteraceae. Shifts in gut microbial communities in response to host blood-meal source may fundamentally impact pathogen transmission given the well-documented link between specific bacterial taxa and vector susceptibility to a variety of mosquito-borne pathogens and may be a key determinant of individual and population variation in vector competence.

Invasive alien plants wreak havoc on native ecosystems and using them as a source of biopesticides could improve their management. We examined the toxicity of essential oil of wild carrot (also known as 'Queen Anne's Lace', Daucus carota Linnaeus), an aggressive invader throughout the United States, against Aedes aegypti L., Culex pipiens L., and Culex restuans Theobald larvae. Comparisons were made between essential oil extracted from umbels of local populations of wild carrot versus a commercial brand. Methyl isoeugenol (60.7%) was by far the most abundant constituent in commercial brand oil, whereas α-pinene (33.0%) and β-pinene (25.8%) were the dominant constituents in essential oil extracted from local wild carrot populations. The commercial brand essential oil was significantly more toxic to Cx. restuans larvae (LC50 = 44.4 ppm) compared with Cx. pipiens (LC50 = 51.0 ppm) and Ae. aegypti (LC50 = 54.5 ppm). Essential oil from local populations of wild carrot was significantly more toxic to both Cx. pipiens (LC50 = 42.9) and Cx. restuans (LC50 = 40.3) larvae compared with Ae. aegypti (LC50 = 64.6 ppm) larvae. Three of the nine tested chemical constituents of wild carrot essential oil (terpinolene, para cymene, and γ-terpinene) were consistently more toxic to larvae of the three mosquito species than the whole essential oil. These findings suggest that exploiting wild carrot essential oil and its chemical constituents as a biopesticide for mosquito control could be used as part of multifaceted approaches for controlling this invasive alien plant species.

Successful infection of the insect body by entomopathogenic fungi is the result of complex molecular interactions between the host and the invading pathogenic fungi. The mosquito antifungal response is multifaceted and is regulated in part by the Toll and Jak-STAT pathways. Here, we assessed the role of the IMD pathway in the mosquito Ae. aegypti antifungal immune response when challenged with one of two entomopathogenic fungi, Beauveria bassiana and Isaria javanica. IMD pathway components of the mosquito immune system were elicited in response to infection with both entomopathogenic fungi, primarily in the fat body of mosquitoes. Furthermore, we observed induction of antimicrobial peptides that in turn appear to be tissue and fungal strain-specific. IMD pathway impairment by RNAi gene silencing resulted in higher fungal proliferation and reduction in survival of fungi-infected mosquitoes. Collectively, these data indicates that the IMD pathway plays a more significant role in the antifungal immune response than previously recognized.

Ecological traps occur due to a mismatch between a habitat's attractiveness and quality, wherein organisms show preference for low-quality habitats over other available high-quality habitats. Our previous research identified leaf litter from common blackberry (Rubus allegheniensis) as a natural ecological trap for an important vector for West Nile virus (Culex pipiens), attracting mosquitoes to oviposit in habitats deleterious to the survival of their larvae. Here we demonstrate that manipulation of leaf litter in stormwater catch basins, an important source of disease vector mosquitoes in urban environments, can increaseCx. pipiensoviposition but reduce survival. In a series of experiments designed to elucidate the mechanisms that explain the attractive and lethal properties of this native plant, behavioural bioassays suggest that oviposition site selection byCx. pipiensis mediated primarily by chemical cues as leaves decompose. However, we also show that juvenile mosquito survival is mainly related to the suitability of the bacterial community in the aquatic habitat for mosquito nutritional needs, which does not appear to create a cue that influences oviposition choice. This mismatch between oviposition cues and drivers of larval habitat quality may account for the ecological trap phenomenon detected in this study. Our findings provide new insights into potential mechanistic pathways by which ecological traps may occur in nature and proof-of-concept for a new ‘attract-and-kill’ tool for mosquito control.

Development of alternative vector control strategies are becoming more pressing given the rapid evolution of insecticide resistance and the rise of vector borne pathogens affecting public health such as dengue, chikungunya and Zika. Fungal-based biopesticides are promising alternatives to synthetic insecticides because they are ecofriendly and are highly effective at infecting insects through contact. This study evaluated the susceptibility of the yellow fever mosquito Ae. aegypti to a range of entomopathogenic fungal strains from the genus Isaria. We observed a diverse variation in the virulence of the Isaria strains tested, with two strains showing high pathogenicity towards adult mosquitoes. Mosquito susceptibility to fungal infection was further corroborated through the molecular quantification of fungal loads and the transcript evaluation of a fungal-specific pathogen recognition molecule in the mosquito body. Moreover, quantitative analysis of transcript abundance coupled with enzymatic assays revealed strain-specific subversion of the melanization cascade, an important immune response component. Our study contributes critical insights for a better understanding of fungal-mosquito interactions.