Long-Term Rodent Surveillance after Outbreak of Hantavirus Infection, Yosemite National Park, California, USA, 2012.

In 2012, a total of 9 cases of hantavirus infection occurred in overnight visitors to Yosemite Valley, Yosemite National Park, California, USA. In the 6 years after the initial outbreak investigation, the California Department of Public Health conducted 11 rodent trapping events in developed areas of Yosemite Valley and 6 in Tuolumne Meadows to monitor the relative abundance of deer mice (Peromyscus maniculatus) and seroprevalence of Sin Nombre orthohantavirus, the causative agent of hantavirus pulmonary syndrome. Deer mouse trap success in Yosemite Valley remained lower than that observed during the 2012 outbreak investigation. Seroprevalence of Sin Nombre orthohantavirus in deer mice during 2013-2018 was also lower than during the outbreak, but the difference was not statistically significant (p = 0.02). The decreased relative abundance of Peromyscus spp. mice in developed areas of Yosemite Valley after the outbreak is probably associated with increased rodent exclusion efforts and decreased peridomestic habitat.

Effect of Holding Conditions on the Detection of Chikungunya and Venezuelan Equine Encephalitis Viruses in Mosquito Pools.

Emerging and re-emerging arboviruses continue to be a threat to global public health, and viral surveillance of mosquito populations is critical for mosquito control operations. Due to the tropical climate of many of the affected areas, it may be difficult to maintain a cold chain as the samples travel from collection sites to laboratories for testing. We determined how suboptimal holding temperatures affected the ability to detect viruses in pools of mosquitoes. Adult female Aedes albopictus and Ae. taeniorhynchus individuals were inoculated with chikungunya virus or Venezuelan equine encephalitis virus suspensions, respectively, and placed at 26°C for 8 days. One infected mosquito was then added to a vial of 24 negative mosquitoes and held at -80°C, -20°C, 4°C, 22°C, or 35°C for up to 14 days. Mosquito pools were analyzed for both infectious virus by plaque assay and for viral ribonucleic acid (RNA) with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). At higher temperatures, the amount of infectious virus decreased rapidly, but viruses in samples held at 4°C or lower remained relatively stable. In contrast, viral RNA was detectable from pools held at all temperatures and holding times by RT-qPCR. Cycle threshold (Ct) values increased as temperatures and holding times increased. These findings suggest that if viral RNA detection is the goal of surveillance efforts, then mosquito pools do not require storage at ≤4°C. This enhances the feasibility of field-based arbovirus surveillance programs in which maintaining a cold chain may not be a possibility.

First Record of Aedes albopictus in Georgia and Updated Checklist of Reported Species.

Mosquito surveillance was carried out in Batumi, Georgia, in August 2014. Aedes albopictus was detected for the first time, which brought the number of reported mosquito species in Georgia to 32. An updated checklist of the mosquitoes of Georgia is provided.

Reactive oxygen species production and Brugia pahangi survivorship in Aedes polynesiensis with artificial Wolbachia infection types.

Heterologous transinfection with the endosymbiotic bacterium Wolbachia has been shown previously to induce pathogen interference phenotypes in mosquito hosts. Here we examine an artificially infected strain of Aedes polynesiensis, the primary vector of Wuchereria bancrofti, which is the causative agent of Lymphatic filariasis (LF) throughout much of the South Pacific. Embryonic microinjection was used to transfer the wAlbB infection from Aedes albopictus into an aposymbiotic strain of Ae. polynesiensis. The resulting strain (designated "MTB") experiences a stable artificial infection with high maternal inheritance. Reciprocal crosses of MTB with naturally infected wild-type Ae. polynesiensis demonstrate strong bidirectional incompatibility. Levels of reactive oxygen species (ROS) in the MTB strain differ significantly relative to that of the wild-type, indicating an impaired ability to regulate oxidative stress. Following a challenge with Brugia pahangi, the number of filarial worms achieving the infective stage is significantly reduced in MTB as compared to the naturally infected and aposymbiotic strains. Survivorship of MTB differed significantly from that of the wild-type, with an interactive effect between survivorship and blood feeding. The results demonstrate a direct correlation between decreased ROS levels and decreased survival of adult female Aedes polynesiensis. The results are discussed in relation to the interaction of Wolbachia with ROS production and antioxidant expression, iron homeostasis and the insect immune system. We discuss the potential applied use of the MTB strain for impacting Ae. polynesiensis populations and strategies for reducing LF incidence in the South Pacific.

Interspecific Transfer of a Wolbachia Infection Into Aedes albopictus (Diptera: Culicidae) Yields a Novel Phenotype Capable of Rescuing a Superinfection.

Wolbachia are maternally transmitted obligate bacteria that occur naturally in many arthropods. The phenotype observed in mosquitoes is known as cytoplasmic incompatibility (CI), which results in reduced or absent egg hatch in crosses between individuals with different infection types. Applied mosquito control strategies propose that by releasing individuals infected with a Wolbachia strain that differs from that in the natural host population, CI could be used to suppress or replace mosquito populations. Here, using tetracycline treatment and embryonic microinjection, Aedes albopictus (Skuse) was cleared of its natural Wolbachia infection and artificially infected with a Wolbachia strain originating from Aedes riversi Bohart & Ingram. Crossing experiments were carried out to determine whether CI could be observed between the artificially infected strain (UC), naturally infected (wild type), and uninfected strains of Ae. albopictus. Crosses between UC males and uninfected females resulted in no egg hatch, a classic unidirectional CI pattern. Crosses between the wild-type and UC strain also exhibited a unidirectional pattern of CI, demonstrating that the UC strain is compatible with both of the Wolbachia types that occur within Ae. albopictus and that wild-type Wolbachia infections are unable to fully rescue the UC Wolbachia type. Crosses between the UC strain and another artificially infected Ae. albopictus strain (ARwP), were bidirectionally incompatible, demonstrating that the UC strain is not compatible with all Wolbachia types. The CI patterns observed in this study were atypical and the opposite of that typically observed with superinfections.

Species Diversity, Seasonal, and Spatial Distribution of Mosquitoes (Diptera: Culicidae) Captured in Aotus Monkey-Baited Traps in a Forested Site Near Iquitos, Peru.

This study was conducted to determine the relative abundance, diversity, seasonal, and vertical distributions of potential mosquito vectors in the Amazon Basin, Peru. A total of 66,097 mosquitoes (50 mosquito species from 12 genera) were collected from May 2001 through March 2002 at a forested site near Iquitos, Peru. Mosquitoes were collected using Aotus nancymae Hershkovitz monkey-baited CDC light traps set for 12-h day and night periods at varying heights (e.g., ground and canopy) in the forest. Of the 12 genera, three accounted for 75% of all mosquitoes collected: Culex (33%), Aedes (23%), and Psorophora (18%). The most prevalent species collected were Aedes serratus (Theobald), Culex pedroi Sirivanakarn & Belkin, Psorophora albigenu (Peryassu), and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker), which accounted for 56% of all mosquitoes captured. In general, mosquitoes were collected more often at night and on the ground. Exceptions include Coquillettidia venezuelensis (Theobald), which were collected in relatively even numbers at both day and night and most Mansonia and some species of Anopheles, which were collected more often in the canopy. Total mosquito populations had two peaks, June-July (Ma. indubitans/titillans and Cq. venezuelensis) and December-January (Ps. albigenu, Cx. pedroi, and Ae. serratus). Observations of the eight most collected mosquitoes indicated that behavioral shifts were not observed between collection months. These data provide a better understanding of the species diversity, population density, and seasonal distribution of potential mosquito vectors within the Amazon Basin region and allow for the development of appropriate vector and disease prevention strategies.

Detection and Isolation of Rickettsia tillamookensis (Rickettsiales: Rickettsiaceae) From Ixodes pacificus (Acari: Ixodidae) From Multiple Regions of California.

The western black-legged tick (Ixodes pacificus) is the most frequently identified human-biting tick species in the western United States and the principal vector of at least three recognized bacterial pathogens of humans. A potentially pathogenic Rickettsia species, first described in 1978 and recently characterized as a novel transitional group agent designated as Rickettsia tillamookensis, also exists among populations of I. pacificus, although the distribution and frequency of this agent are poorly known. We evaluated DNA extracts from 348 host-seeking I. pacificus nymphs collected from 9 locations in five California counties, and from 916 I. pacificus adults collected from 24 locations in 13 counties, by using a real-time PCR designed specifically to detect DNA of R. tillamookensis. DNA of R. tillamookensis was detected in 10 (2.9%) nymphs (95% CI: 1.6-5.2%) and 17 (1.9%) adults (95% CI: 1.2-3.0%) from 11 counties of northern California. Although site-specific infection rates varied greatly, frequencies of infection remained consistently low when aggregated by stage, sex, habitat type, or geographical region. Four novel isolates of R. tillamookensis were cultivated in Vero E6 cells from individual adult ticks collected from Alameda, Nevada, and Yolo counties. Four historical isolates, serotyped previously as 'Tillamook-like' strains over 40 yr ago, were revived from long-term storage in liquid nitrogen and confirmed subsequently by molecular methods as isolates of R. tillamookensis. The potential public health impact of R. tillamookensis requires further investigation.

Pre-existing Microfilarial Infections of American Robins (Passeriformes: Turdidae) and Common Grackles (Passeriformes: Icteridae) Have Limited Impact on Enhancing Dissemination of West Nile Virus in Culex pipiens Mosquitoes (Diptera: Culicidae).

Microfilariae (MF) are the immature stages of filarial nematode parasites and inhabit the blood and dermis of all classes of vertebrates, except fish. Concurrent ingestion of MF and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Shortly after being ingested, MF penetrate the mosquito's midgut and may introduce virus into the mosquito's hemocoel, creating a disseminated viral infection much sooner than normal. This phenomenon is known as microfilarial enhancement. Both American Robins and Common Grackles harbor MF-that is, Eufilaria sp. and Chandlerella quiscali von Linstow (Spirurida: Onchocercidae), respectively. We compared infection and dissemination rates in Culex pipiens L. mosquitoes that fed on birds with and without MF infections that had been infected with West Nile virus (WNV). At moderate viremias, about 107 plaque-forming units (pfu)/ml of blood, there were no differences in infection or dissemination rates among mosquitoes that ingested viremic blood from a bird with or without microfilaremia. At high viremias, >108.5 pfu/ml, mosquitoes feeding on a microfilaremic Grackle with concurrent viremia had significantly higher infection and dissemination rates than mosquitoes fed on viremic Grackles without microfilaremia. Microfilarial enhancement depends on the specific virus, MF, and mosquito species examined. How virus is introduced into the hemocoel by MF differs between the avian/WNV systems described here (i.e., leakage) and various arboviruses with MF of the human filarid, Brugia malayi (Brug) (Spirurida: Onchocercidae) (i.e., cotransport). Additional studies are needed to determine if other avian species and their MF are involved in the microfilarial enhancement of WNV in nature.

A Comparison of Questing Substrates and Environmental Factors That Influence Nymphal Ixodes pacificus (Acari: Ixodidae) Abundance and Seasonality in the Sierra Nevada Foothills of California.

In California, the western blacklegged tick, Ixodes pacificus Cooley and Kohls, is the principal vector of the Borrelia burgdorferi sensu lato (sl) complex (Spirochaetales: Spirochaetaceae, Johnson et al.), which includes the causative agent of Lyme disease (B. burgdorferi sensu stricto). Ixodes pacificus nymphs were sampled from 2015 to 2017 at one Sierra Nevada foothill site to evaluate our efficiency in collecting this life stage, characterize nymphal seasonality, and identify environmental factors affecting their abundance and infection with B. burgdorferi sl. To assess sampling success, we compared the density and prevalence of I. pacificus nymphs flagged from four questing substrates (logs, rocks, tree trunks, leaf litter). Habitat characteristics (e.g., canopy cover, tree species) were recorded for each sample, and temperature and relative humidity were measured hourly at one location. Generalized linear mixed models were used to assess environmental factors associated with I. pacificus abundance and B. burgdorferi sl infection. In total, 2,033 substrates were sampled, resulting in the collection of 742 I. pacificus nymphs. Seasonal abundance of nymphs was bimodal with peak activity occurring from late March through April and a secondary peak in June. Substrate type, collection year, month, and canopy cover were all significant predictors of nymphal density and prevalence. Logs, rocks, and tree trunks had significantly greater nymphal densities and prevalences than leaf litter. Cumulative annual vapor pressure deficit was the only significant climatic predictor of overall nymphal I. pacificus density and prevalence. No associations were observed between the presence of B. burgdorferi sl in nymphs and environmental variables.

Human-Biting Ixodes Ticks and Pathogen Prevalence from California, Oregon, and Washington.

From July 2006 through August 2017, a passive surveillance study of Ixodes ticks submitted from California, Oregon, and Washington was conducted by the TickReport program at the University of Massachusetts, Amherst. In total, 549 human-biting Ixodes ticks were submitted comprising both endemic and nonendemic species. We found that 430 endemic ticks were from 3 Ixodes species: Ixodes pacificus, Ixodes spinipalpis, and Ixodes angustus, whereas Ixodes scapularis (n = 111) was the most common species among the 119 nonendemic ticks. The submission peak for nymphal I. pacificus and I. spinipalpis was June, while submission peak for adult I. pacificus and nymphal I. angustus was April and September, respectively. Endemic ticks commonly attached to the lower extremities of their victims, and individuals younger than 9 years old were frequently bitten. The infection prevalence of Borrelia burgdorferi sensu lato, Borrelia miyamotoi, and Anaplasma phagocytophilum in I. pacificus ticks was 1.31%, 1.05%, and 0.52%, respectively, and the prevalence of B. burgdorferi s. l. and A. phagocytophilum in I. spinipalpis ticks was 14.29% and 10.71%, respectively. Furthermore, two species within the B. burgdorferi s. l. complex were detected in West Coast ticks: B. burgdorferi sensu stricto and Borrelia lanei. I. spinipalpis had the highest Borrelia prevalence among endemic ticks, and it was caused exclusively by B. lanei. Borrelia mayonii, Babesia microti, and Ehrlichia muris-like agent were not detected in these endemic ticks. In this study, we show that many nonendemic Ixodes ticks (119/549) are most likely acquired from travel to a different geographic region. We report cases of conventionally recognized nonhuman feeders (I. spinipalpis and I. angustus) parasitizing humans. The highest pathogen prevalence in I. spinipalpis may indicate a larger public health threat than previously thought, and the enzootic life cycle and pathogenicity of B. lanei warrant further study.

Wolbachia effects on Rift Valley fever virus infection in Culex tarsalis mosquitoes.

Innovative tools are needed to alleviate the burden of mosquito-borne diseases, and strategies that target the pathogen are being considered. A possible tactic is the use of Wolbachia, a maternally inherited, endosymbiotic bacterium that can (but does not always) suppress diverse pathogens when introduced to naive mosquito species. We investigated effects of somatic Wolbachia (strain wAlbB) infection on Rift Valley fever virus (RVFV) in Culex tarsalis mosquitoes. When compared to Wolbachia-uninfected mosquitoes, there was no significant effect of Wolbachia infection on RVFV infection, dissemination, or transmission frequencies, nor on viral body or saliva titers. Within Wolbachia-infected mosquitoes, there was a modest negative correlation between RVFV body titers and Wolbachia density, suggesting that Wolbachia may slightly suppress RVFV in a density-dependent manner in this mosquito species. These results are contrary to previous work in the same mosquito species, showing Wolbachia-induced enhancement of West Nile virus infection rates. Taken together, these results highlight the importance of exploring the breadth of pathogen modulations induced by Wolbachia.

Analyzing arthropods for the presence of bacteria.

Bacteria within arthropods can be identified using culture-independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplification using 16S rDNA general bacteria primers, and profiling the bacterial community using denaturing gradient gel electrophoresis.

Pollinator and herbivore attraction to cucurbita floral volatiles.

Mutualists and antagonists may place conflicting selection pressures on plant traits. For example, the evolution of floral traits is typically studied in the context of attracting pollinators, but traits may incur fitness costs if they are also attractive to antagonists. Striped cucumber beetles (Acalymma vittatum) feed on cucurbits and are attracted to several volatiles emitted by Cucurbita blossoms. However, the effect of these volatiles on pollinator attraction is unknown. Our goal was to determine whether pollinators were attracted to the same or different floral volatiles as herbivorous cucumber beetles. We tested three volatiles previously found to attract cucumber beetles in a factorial design to determine attraction of squash bees (Peponapis pruinosa), the specialist pollinators of cucurbita species, as well as the specialist herbivore A. vittatum. We found that 1,2,4-trimethoxybenzene was attractive to both the pollinator and the herbivore, indole was attractive only to the herbivore, and (E)-cinnamaldehyde was attractive only to the pollinator. There were no interactions among volatiles on attraction of squash bees or cucumber beetles. Our results suggest that reduced indole emission could benefit plants by reducing herbivore attraction without loss of pollination, and that 1,2,4-trimethoxybenzene might be under conflicting selection pressure from mutualists and antagonists. By examining the attraction of both mutualists and antagonists to Cucurbita floral volatiles, we have demonstrated the potential for some compounds to influence only one type of interaction, while others may affect both interactions and possibly result in tradeoffs. These results shed light on the potential evolution of fragrance in native Cucurbita, and may have consequences for yield in agricultural settings.