Back to the Future: Quantifying Wing Wear as a Method to Measure Mosquito Age.

Vector biologists have long sought the ability to accurately quantify the age of wild mosquito populations, a metric used to measure vector control efficiency. This has proven difficult due to the difficulties of working in the field and the biological complexities of wild mosquitoes. Ideal age grading techniques must overcome both challenges while also providing epidemiologically relevant age measurements. Given these requirements, the Detinova parity technique, which estimates age from the mosquito ovary and tracheole skein morphology, has been most often used for mosquito age grading despite significant limitations, including being based solely on the physiology of ovarian development. Here, we have developed a modernized version of the original mosquito aging method that evaluated wing wear, expanding it to estimate mosquito chronological age from wing scale loss. We conducted laboratory experiments using adult Anopheles gambiae held in insectary cages or mesocosms, the latter of which also featured ivermectin bloodmeal treatments to change the population age structure. Mosquitoes were age graded by parity assessments and both human- and computational-based wing evaluations. Although the Detinova technique was not able to detect differences in age population structure between treated and control mesocosms, significant differences were apparent using the wing scale technique. Analysis of wing images using averaged left- and right-wing pixel intensity scores predicted mosquito age at high accuracy (overall test accuracy: 83.4%, average training accuracy: 89.7%). This suggests that this technique could be an accurate and practical tool for mosquito age grading though further evaluation in wild mosquito populations is required.

Development of a real-time PCR assay to detect the single nucleotide polymorphism causing Warmblood Fragile Foal Syndrome.

Warmblood Fragile Foal syndrome (WFFS) is an autosomal recessive condition that affects the maturation of collagen in affected foals. Foals affected with the disease typically die or are euthanised shortly after birth. WFFS is caused by a single nucleotide change at position 2032 of the equine PLOD1 gene, causing an impairment of the wild-type enzyme. A commercial test for the causative genetic mutation is currently available from companies operating under licence from Cornell University but it has limitations. This test requires amplification of a region of the PLOD1 gene encompassing the site of interest, followed by Sanger sequencing of that region and computational analysis. We describe here the development of an alternative, real-time PCR based assay that rapidly and reliably differentiates between the wild-type and WFFS associated nucleotides without the need for sequencing, thus increasing the potential for high throughput analysis of large numbers of samples in a cost-effective manner.

Three Immunocompetent Small Animal Models That Do Not Support Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-borne flavivirus that is primarily transmitted to humans through the bite of an infected mosquito. ZIKV causes disease in infected humans with added complications of Guillain-Barré syndrome and birth defects in infants born to mothers infected during pregnancy. There are several large immunocompetent animal models for ZIKV including non-human primates (NHPs). NHP models closely reflect human infection; however, due to sample size restrictions, investigations into the effects of transmission route and the impacts on disease dynamics have been understudied. Mice have been widely used for modeling ZIKV infection, yet there are few ZIKV-susceptible immunocompetent mouse models and none of these have been used to investigate sexual transmission. In an effort to identify a small immunocompetent animal model to characterize sexual transmission of ZIKV, we attempt experimental infection of multimammate mice, New Zealand white rabbits, and Hartley guinea pigs. The multimammate mouse is the natural reservoir of Lassa fever virus and has been identified to harbor other human pathogens. Likewise, while NZW rabbits are susceptible to West Nile virus, they have not yet been examined for their susceptibility to infection with ZIKV. Guinea pigs have been successfully used as models for ZIKV infection, but only in immunocompromised life stages (young or pregnant). Here, it was found that the multimammate mouse and New Zealand White (NZW) rabbits are not susceptible ZIKV infection as determined by a lack viral RNA in tissues and fluids collected. Sexually mature male Hartley guinea pigs were inoculated subcutaneously and by mosquito bite, but found to be refractory to ZIKV infection, contrary to findings of other studies in young and pregnant guinea pigs. Interestingly, here it is shown that adult male guinea pigs are not susceptible to ZIKV infection, even when infected by natural route (e.g., mosquito bite). Although a new small animal model for the sexual transmission for ZIKV was not established through this study, these findings provide information on outbred animal species that are not permissive to infection (NZW rabbits and multimammate mice) and new information surrounding limitations of a previously established animal model (guinea pigs).

Characterizing and Quantifying Arbovirus Transmission by Aedes aegypti Using Forced Salivation and Analysis of Bloodmeals.

Arbovirus transmission studies are dependent on the ability to estimate the titer of virus transmitted from infectious mosquitoes to a host. There are several methods for estimating virus titer in mosquito saliva, including (1) using forced salivation (FS) whereby the infectious mosquito's proboscis is forced into a capillary tube containing media to collect and test their saliva for virus, and (2) by quantifying virus expectorated into host tissues or into the blood contained in an artificial feeder immediately after blood feeding. We studied FS and bloodmeals to estimate and compare titers of Zika virus and chikungunya virus transmitted by the mosquito vector Aedes aegypti. Infectious virus and viral genomes of both viruses were detected more often from individual mosquitoes using immersion oil for the FS media compared to fetal bovine serum (FBS) plus glycerol, but the FS media had no influence on virus quantification from positive samples. FS virus titers were equivalent when comparing individuals or groups of mosquitoes that never received a blood meal compared to those that were blood fed immediately prior, showing that blood feeding does not influence FS. This suggested that performing FS on mosquitoes after blood feeding might be an efficient way to estimate virus transmitted during blood feeding. However, detecting virus from the blood remaining in an artificial feeder post-blood feeding was mostly unsuccessful relative to quantifying virus from FS of the post-blood fed mosquitoes. In contrast, immunocompromised mice always became infected after being fed on by Zika-infected mosquitoes, even when no infectious virus was detected in their saliva by FS post-blood feed. Due to this discrepancy, we tested the ingested bloodmeals of individual mosquitoes that fed on artificial blood feeders for virus, and compared these to virus in their saliva harvested from FS and to virus in their bodies. These experiments revealed ~50-100 times higher virus titers in the dissected bloodmeals compared to those detected in the same mosquitoes' saliva, demonstrating how mosquitoes re-ingest much of their saliva during artificial blood feeding, and highlighting a large increase in virus transmission during Aedes aegypti blood feeding. Both FS and the dissected bloodmeals of artificially blood-fed mosquitoes showed that the quantity of viral RNA expectorated by mosquitoes was 2-5 logs more than the quantity of infectious virus. The results from this study add critical information to understanding and quantifying the transmission of Aedes aegypti arboviruses.

Nootkatone Is an Effective Repellent against Aedes aegypti and Aedes albopictus.

We tested a nootkatone product for insecticide activity against the most prominent vectors of Zika virus (ZIKV), Aedes aegypti, and Aedes albopictus. We tested the permethrin-resistant (PERM-R) Vergel strain of A. aegypti and the permethrin-susceptible (PERM-S) New Orleans strain of A. aegypti to determine if insecticide resistance affected their susceptibility to nootkatone. Bottle bioassays showed that the PERM-S strain (New Orleans) was more susceptible to nootkatone than the confirmed A. aegypti permethrin-resistant (PERM-R) strain, Vergel. The A. albopictus strain ATM-NJ95 was a known PERM-S strain and Coatzacoalcos permethrin susceptibility was unknown but proved to be similar to the ATM-NJ95 PERM-S phenotype. The A. albopictus strains (ATM-NJ95 and Coatzacoalcos) were as susceptible to nootkatone as the New Orleans strain. Bottle bioassays conducted with ZIKV-infected mosquitoes showed that the New Orleans (PERM-S) strain was as susceptible to nootkatone as the mock-infected controls, but the PERM-R strain was less susceptible to nootkatone than the mock-infected controls. Repellency/irritancy and biting inhibition bioassays (RIBB) of A. aegypti determined whether the nootkatone-treated arms of three human subjects prevented uninfected A. aegypti mosquitoes from being attracted to the test subjects and blood-feeding on them. The RIBB analyses data calculated the spatial activity index (SAI) and biting inhibition factor (BI) of A. aegypti at different nootkatone concentrations and then compared the SAI and BI of existing repellency products. We concluded that nootkatone repelled mosquitoes at a rate comparable to 7% DEET or 5% picaridin and has the potential to be an efficacious repellent against adult A. aegypti mosquitoes.

A comparison of the effect of weather and climate on emergency department visitation in Roanoke and Charlottesville, Virginia.

Compared with mortality, the impact of weather and climate on human morbidity is less well understood, especially in the cold season. We examined the relationships between weather and emergency department (ED) visitation at hospitals in Roanoke and Charlottesville, Virginia, two locations with similar climates and population demographic profiles. Using patient-level data obtained from electronic medical records, each patient who visited the ED was linked to that day's weather from one of 8 weather stations in the region based on each patient's ZIP code of residence. The resulting 2010-2017 daily ED visit time series were examined using a distributed lag non-linear model to account for the concurrent and lagged effects of weather. Total ED visits were modeled separately for each location along with subsets based on gender, race, and age. The relationship between the relative risk of ED visitation and temperature or apparent temperature over lags of one week was positive and approximately linear at both locations. The relative risk increased about 5% on warm, humid days in both cities (lag 0 or lag 1). Cold conditions had a protective effect, with up to a 15% decline on cold days, but ED visits increased by 4% from 2 to 5 days after the cold event. The effect of thermal extremes tended to be larger for non-whites and the elderly, and there was some evidence of a greater lagged response for non-whites in Roanoke. Females in Roanoke were more impacted by winter cold conditions than males, who were more likely to show a lagged response at high temperatures. In Charlottesville, males sought ED attention at lower temperatures than did females. The similarities in the ED response patterns between these two hospitals suggest that certain aspects of the response may be generalizable to other locations that have similar climates and demographic profiles.