A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes.

BACKGROUND & OBJECTIVES: Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. METHODS: Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. RESULTS: In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x104 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100. In full combination assays, CHIKV was detected to 1.32x104, DENV1-4 to 3.79x101 and ZIKV to 1.07x100. INTERPRETATION & CONCLUSION: This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

Clusters of bioactive compounds target dynamic endomembrane networks in vivo.

Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana. Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.

Small RNA sequencing of field Culex mosquitoes identifies patterns of viral infection and the mosquito immune response.

Mosquito-borne disease remains a significant burden on global health. In the United States, the major threat posed by mosquitoes is transmission of arboviruses, including West Nile virus by mosquitoes of the Culex genus. Virus metagenomic analysis of mosquito small RNA using deep sequencing and advanced bioinformatic tools enables the rapid detection of viruses and other infecting organisms, both pathogenic and non-pathogenic to humans, without any precedent knowledge. In this study, we sequenced small RNA samples from over 60 pools of Culex mosquitoes from two major areas of Southern California from 2017 to 2019 to elucidate the virome and immune responses of Culex. Our results demonstrated that small RNAs not only allowed the detection of viruses but also revealed distinct patterns of viral infection based on location, Culex species, and time. We also identified miRNAs that are most likely involved in Culex immune responses to viruses and Wolbachia bacteria, and show the utility of using small RNA to detect antiviral immune pathways including piRNAs against some pathogens. Collectively, these findings show that deep sequencing of small RNA can be used for virus discovery and surveillance. One could also conceive that such work could be accomplished in various locations across the world and over time to better understand patterns of mosquito infection and immune response to many vector-borne diseases in field samples.

Characterization of the Blood-Feeding Patterns of Culex quinquefasciatus (Diptera: Culicidae) in San Bernardino County, California.

West Nile virus (WNV) is a zoonotic disease that is endemic in North America and is known to cause a range of symptoms from mild to life threatening in humans. Culex quinquefasciatus is one of the most prominent vectors of WNV in Southern California. The goal of this study was to identify which animal species are most fed upon by these mosquitoes in various habitats in the West Valley area of San Bernardino County, California, and determine the relationship between blood-feeding patterns and WNV activity in the region. Culex quinquefasciatus specimens were collected by West Valley Mosquito and Vector Control District during 2011 from 32 different sites. The bloodmeals of 683 individuals (92.4% of those tested) were identified using the mitochondrial gene cytochrome c oxidase 1 (COI). These bloodmeals comprised 29 vertebrate species across four different habitats. Species richness (ranging from 10 to 17) was not significantly different between habitats when rarified to account for sample size. Across habitats, the highest percentage of avian bloodmeals were taken from house sparrows (18.8-39.1%) and house finches (2.6-31.5%). Bloodmeals were identified from five mammalian species, accounting for 5.1-59.2% of bloodmeals by habitat, including humans (0-4.1%). A seasonal shift towards increased mammalian bloodmeal prevalence, specifically for domestic dog and human bloodmeals, was observed in urban habitats. The WNV activity during 2011 in San Bernardino County occurred mostly in urban and suburban areas as indicated by minimum infection rate (MIR) in Culex quinquefasciatus, notable as all human bloodmeals were identified from these two habitats.

Establishment of the Invasive Aedes aegypti (Diptera: Culicidae) in the West Valley Area of San Bernardino County, CA.

The yellow fever mosquito, Aedes aegypti (Linnaeus, 1762), is the most aggressive invasive mosquito species with worldwide distribution. In addition to being a notorious nuisance species, it can pose significant public health concern because of its ability to transmit various viral pathogens. The first adult capture in the West Valley area of San Bernardino County, CA, occurred in September 2015 in Montclair. A strategic surveillance plan was implemented accordingly by the West Valley Mosquito and Vector Control District to document the infestation. The Biogent Sentinel (BG-2) trap augmented with BG-Lure and carbon dioxide (CO2) was deployed as a routine surveillance tool during 2017-2019. Extensive trapping revealed an expanding infestation, when positive trap nights (TN) increased from 14.2% in 2017 to 23.9% in 2018 and 55.6% in 2019. The average counts/TN increased from 0.65 in 2017 and 0.90 in 2018 to 3.83 in 2019. The cities of Montclair, Chino, and Ontario had much higher infestation than other cities in the district with the highest positive TN of 46.0% in Montclair, and highest average trap count of 3.23/TN in Chino. It was interesting to note that males coincided with females with more profound trend during warmer months of July to October when ratios of males ranged 28.4-35.0%. The BG-2 trap significantly outperformed the CO2 trap and gravid trap. The establishment of this invasive species in semiarid inland Southern California was further confirmed by concurrent larval collections.

Cross Resistance in S-Methoprene-Resistant Culex quinquefasciatus (Diptera: Culicidae).

The juvenile hormone analog S-methoprene is the only synthetic biopesticide that is registered with the United States Environmental Protection Agency to control arthropods of economic importance in public health, livestock, pets, urban, and stored products. The high activity, relative target specificity, and benign environmental profile of S-methoprene have been well documented. While the risk of resistance in mosquitoes to S-methoprene is generally low, there is a lack of information regarding cross resistance in S-methoprene-resistant mosquitoes to other pesticides. In this paper, a population of the southern house mosquito Culex quinquefasciatus Say from southern California acquired low levels of resistance to S-methoprene in the field, where the resistance ratios ranged 7.0- to 8.8-fold as compared with a laboratory reference colony. After 30 generations of laboratory selections by S-methoprene when resistance was elevated to 57.4- to 168.3-fold relative to an unselected population, various levels of cross resistance to other commonly used pesticides were revealed in the selected population. Cross resistance to the microbial mosquito larvicide Lysinibacillus sphaericus (Meyer & Neide) (Bacillales: Bacillaceae) was the most profound, amounting to 77.50- to 220.50-fold. The mechanism and potential management tactics toward cross resistance are discussed to preserve the unique value of this synthetic biopesticide.

Deployment and Fact Analysis of the In2Care® Mosquito Trap, A Novel Tool for Controlling Invasive Aedes Species.

During April-October 2019, the West Valley Mosquito and Vector Control District (Ontario, CA) deployed large numbers of In2Care® mosquito traps in a preliminary study to evaluate the trap's potential effectiveness at controlling invasive Aedes aegypti (L.) and Ae. albopictus (Skuse) in 6 cities of San Bernardino County, CA. The trap was used to attract ovipositing females, expose them to the juvenile hormone mimic pyriproxyfen and the entomopathogenic fungus Beauveria bassiana, and autodisseminate pyriproxyfen to other water sources prior to their death from fungal infection. The trap attracted Ae. aegypti and Culex quinquefasciatus, with the latter species predominating at much higher larval densities in the trap reservoirs. Field-collected larvae and pupae from the trap reservoirs showed complete adult emergence inhibition. Furthermore, the trap reservoirs retained high levels of residual larvicidal, pupicidal, and emergence inhibition activity after they were retrieved from the field, as indicated by laboratory bioassays against laboratory colony of Cx. quinquefasciatus. Results of this study support more detailed quantitative local evaluations on trap efficacy to measure the impact of the In2Care mosquito trap on wild invasive Aedes and Culex populations in future mosquito control efforts.

Susceptibility Profile of Aedes aegypti L. (Diptera: Culicidae) from Montclair, California, to Commonly Used Pesticides, With Note on Resistance to Pyriproxyfen.

The peridomestic anthropophilic Aedes aegypti L. (Diptera: Culicidae) is originated from the wild zoophilic subspecies Aedes aegypti formosus in sub-Saharan Africa, and currently has a broad distribution in human-modified environments of the tropics and subtropics worldwide. In California, breeding populations were initially detected in 2013 in the cities of Fresno, Madera, and San Mateo, and now can be found in 188 cities of 12 counties in the state. Recent genetic studies suggest that this species invaded California on multiple occasions from several regions of the United States and northern Mexico prior to initial detection. As an invasive species and vector for numerous arboviruses, Ae. aegypti is a primary target of surveillance and control in California. In southern California city of Montclair, a population was identified in September 2015, from which a short-term colony was established in an insectary. The susceptibility of this field population to commonly used pesticides with various modes of action, including 15 formulations against larvae and four against adults, was determined, in reference to a susceptible laboratory colony of the same species. No resistance was shown to most pesticides tested. However, tolerance or reduced susceptibility to spinosad, spinetoram, diflubezuron, and fipronil was detected, and modest levels of resistance to pyriproxyfen (resistance ratio = 38.7-fold at IE50 and 81.5-fold at IE90) was observed. Results are discussed based on the field usage and modes of action of the pesticides tested. Strategic selection and application of pesticides against this population of Ae. aegypti in the urban environments should be taken into consideration.

High Resistance to Bacillus sphaericus and Susceptibility to Other Common Pesticides in Culex pipiens (Diptera: Culicidae) from Salt Lake City, UT.

Biorational mosquito larvicides based on microbial organisms and insect growth regulators (IGRs) have played a vital role in integrated mosquito control, particularly since the invasion of West Nile virus to the United States in 1999. Products that are formulated with technical powder of the bacterium, Bacillus sphaericus Neide (recently Lysinibacillus sphaericus Meyer and Neide), are among the ones that have been extensively applied to combat Culex and other mosquito species. Due to the simplicity of the binary toxins, resistance to this pesticide in laboratory and field populations of Culex pipiens L. complex has occurred globally since 1994. A Cx. pipiens population with a high level of resistance to B. sphaericus (VectoLex WDG) was identified in Salt Lake City, UT, in September 2016. The resistance ratios in this population were 20,780.0- and 23,926.9-fold at LC50 and LC90, respectively, when compared with a susceptible population of a laboratory reference colony of the same species. This B. sphaericus-resistant population remained mostly susceptible to other commonly used pesticides to control arthropods of public health and urban significance, including ones based on microbial organisms (Bacillus thuringiensis subsp. israelensis, spinosad, spinetoram, abamectin), IGRs (pyriproxyfen, methoprene, diflubenzuron, novaluron), organophosphate (temephos), neonicotinoid (imidacloprid), phenylpyrazole (fipronil), oxadiazine (indoxacarb), and pyrethroid (permethrin). Results are discussed according to the modes of action of the pesticides tested, and suggestions are made to manage B. sphaericus-resistant mosquito populations.

A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes

Background & objectives: Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. Methods: Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. Results: In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x10-1 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV. When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV. All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100 . In full combination assays, CHIKV was detected to 1.32x10-1, DENV1-4 to 3.79x101 and ZIKV to 1.07x100 . Interpretation & conclusion: This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

High-throughput identification of chemical endomembrane cycling disruptors utilizing tobacco pollen.

Endomembrane cycling processes in plants remain mostly intractable through classical genetic interrogation. Chemical disruption of these processes provides an opportunity to slow or inhibit these processes for study. Tobacco pollen, which is dependent upon endomembrane cycling for tube growth, provides a plant system that is amenable to high-throughput screening of chemical disruptors. We describe here the process that allowed the identification of over 360 endomembrane cycling disruptors.

An approach to quantify endomembrane dynamics in pollen utilizing bioactive chemicals.

Tip growth of pollen tubes and root hairs occurs via rapid polar growth. These rapidly elongating cells require tip-focused endomembrane trafficking for the deposition and recycling of proteins, membranes, and cell wall materials. Most of the image-based data published to date are subjective and non-quantified. Quantitative and comparative descriptors of these highly dynamic processes have been a major challenge, but are highly desirable for genetic and chemical genomics approaches to dissect this biological network. To address this problem, we screened for small molecules that perturbed the localization of a marker for the Golgi Ras-like monomeric G-protein RAB2:GFP expressed in transgenic tobacco pollen. Semi-automated high-throughput imaging and image analysis resulted in the identification of novel compounds that altered pollen tube development and endomembrane trafficking. Six compounds that caused mislocalization and varying degrees of altered movement of RAB2:GFP-labeled endomembrane bodies were used to generate a training set of image data from which to quantify vesicle dynamics. The area, velocity, straightness, and intensity of each body were quantified using semi-automated image analysis tools revealing quantitative differences in the phenotype caused by each compound. A score was then given to each compound enabling quantitative comparisons between compounds. Our results demonstrate that image analysis can be used to quantitatively evaluate dynamic subcellular endomembrane phenotypes induced by bioactive chemicals, mutations, or other perturbing agents as part of a strategy to quantitatively dissect the endomembrane network.

Sortin1-hypersensitive mutants link vacuolar-trafficking defects and flavonoid metabolism in Arabidopsis vegetative tissues.

Sortin1 is a chemical genetic-hit molecule that causes specific mislocalization of plant and yeast-soluble and membrane vacuolar markers. To better understand its mode of action, we designed a Sortin1-hypersensitive screen and identified several Sortin1-hypersensitive and flavonoid-defective mutants. Mechanistically, Sortin1 mimics the effect of the glutathione inhibitor buthionine sulfoximine and alters the vacuolar accumulation of flavonoids, likely blocking their transport through vacuole-localized ABC transporters. Structure-activity relationship studies conducted in Arabidopsis revealed the structural requirements for Sortin1 bioactivity and demonstrated that overlapping Sortin1 substructures can be used to discriminate between vacuolar-flavonoid accumulations and vacuolar-biogenesis defects. We conclude that Sortin1 is a valuable probe for dissecting novel links among flavonoid transport, vacuolar integrity, and the trafficking of vacuolar targeted cargoes in Arabidopsis.