Sediment-Water Interactions Affecting Dissolved-Mercury Distributions in Camp Far West Reservoir, California

Field and laboratory studies were conducted in April and November 2002 to provide the first direct measurements of the benthic flux of dissolved (0.2-micrometer filtered) mercury species (total and methylated forms) between the bottom sediment and water column at three sampling locations within Camp Far West Reservoir, California: one near the Bear River inlet to the reservoir, a second at a mid-reservoir site of comparable depth to the inlet site, and the third at the deepest position in the reservoir near the dam (herein referred to as the inlet, midreservoir and near-dam sites, respectively; Background, Fig. 1). Because of interest in the effects of historic hydraulic mining and ore processing in the Sierra Nevada foothills just upstream of the reservoir, dissolved-mercury species and predominant ligands that often control the mercury speciation (represented by dissolved organic carbon, and sulfides) were the solutes of primary interest. Benthic flux, sometimes referred to as internal recycling, represents the transport of dissolved chemical species between the water column and the underlying sediment. Because of the affinity of mercury to adsorb onto particle surfaces and to form insoluble precipitates (particularly with sulfides), the mass transport of mercury in mining-affected watersheds is typically particle dominated. As these enriched particles accumulate at depositional sites such as reservoirs, benthic processes facilitate the repartitioning, transformation, and transport of mercury in dissolved, biologically reactive forms (dissolved methylmercury being the most bioavailable for trophic transfer). These are the forms of mercury examined in this study. In contrast to typical scientific manuscripts, this report is formatted in a pyramid-like structure to serve the needs of diverse groups who may be interested in reviewing or acquiring information at various levels of technical detail (Appendix 1). The report enables quick transitions between the initial summary information (figuratively at the top of the pyramid) and the later details of methods or results (figuratively towards the base of the pyramid) using hyperlinks to supporting figures and tables, and an electronically linked Table of Contents. During two sampling events, two replicate sediment cores (Coring methods; Fig. 2) from each of three reservoir locations (Fig. 1) were used in incubation experiments to provide flux estimates and benthic biological characterizations. Incubation of these cores provided “snapshots” of solute flux across the sediment-water interface in the reservoir, under benthic, environmental conditions representative of the time and place of collection. Ancillary data, including nutrient and ligand fluxes, were gathered to provide a water-quality framework from which to compare the results for mercury

Phenoloxidase activity acts as a mosquito innate immune response against infection with semliki forest virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses

Transcriptomic and proteomic analysis of arbovirus-infected tick cells

Ticks are important vectors of a wide variety of pathogens including protozoa, bacteria and viruses. Many of the viruses transmitted by ticks are of medical or veterinary importance including tick-borne encephalitis virus (TBEV) and Crimean- Congo hemorrhagic fever virus causing disease in humans, and African swine fever virus and Nairobi sheep disease virus affecting livestock. Although several studies have elucidated tick antimicrobial mechanisms including cellular immune responses such as nodulation, encapsulation and phagocytosis and humoral immune responses such as the JAK/STAT pathway, complement-like proteins, antimicrobial peptides, lectin like pattern-recognition molecules and lysozymes, very little is known about the innate immune response of ticks towards viral infection. This study therefore aimed to identify molecules that might be involved in the response of ticks to viral infection. The hypothesis was that TBEV infection leads to changes in the expression of immunity-related transcripts and proteins in Ixodes spp. tick cells and that at least some of these might be antiviral. Ixodes scapularis-derived cell lines IDE8 and ISE6 were chosen since I. scapularis is currently the only tick species with a sequenced genome and an Ixodes ricinus-derived cell line, IRE/CTVM19, was used because I. ricinus is the natural vector of TBEV. Basic parameters required to study the responses of tick cells to infection were determined, including levels of virus infection, kinetics of virus replication and production, formation of replication complexes and uptake of dsRNA or siRNA. The cell lines IDE8, ISE6 and IRE/CTVM19 were infected with either of two tick-borne flaviviruses, TBEV and Langat virus (LGTV), or with the mosquito-borne alphavirus Semliki Forest virus (SFV). Infection was characterised using techniques including plaque assay, luciferase assay, immunostaining and conventional, confocal and electron microscopy. Two time points for transcriptomics and proteomics analysis of TBEVinfected IDE8 and IRE/CTVM19 cells were selected: day 2 post-infection (p.i.) when virus production was increasing and day 6 p.i. when virus production was decreasing. RNA and protein were isolated from TBEV-infected and mock-infected tick cells at days 2 and 6 p.i. and RNA-Seq and mass spectrometric technologies were used to identify changes in, respectively, transcript and protein abundance. Differential expression of transcripts was determined using the data analysis package DESeq resulting in a total of 43 statistically significantly differentially expressed transcripts in IDE8 cells and 83 in IRE/CTVM19 cells, while differential protein representation using Χ2 test statistics with Bonferroni correction in IDEG6 software resulted in 76 differentially represented proteins in IDE8 cells and 129 in IRE/CTVM19 cells. These included transcripts and proteins which could affect stages of the virus infection, including virus entry, replication, maturation and protein trafficking, and also innate immune responses such as phagocytosis, RNA interference (RNAi), the complement system, the ubiquitin-proteasome pathway, cell stress and the endoplasmic reticulum (ER) stress response. After verification of sequencing data by qRT-PCR, the ability of several of the identified transcripts or proteins to affect virus infection was determined by knockdown experiments in IDE8 and IRE/CTVM19 cells using wild type LGTV, LGTV replicons or TBEV replicons. Knockdown of genes encoding proteins including the ER chaperone gp96 and the heat-shock protein HSP90 resulted in increased virus production in both cell lines, hinting at an antiviral role. In contrast, knockdown of calreticulin, another ER chaperone, resulted in a decrease in virus production in IRE/CTVM19 cells but not in IDE8 cells, implying a requirement for virus production. This functional genomics approach has identified possible novel genes/proteins involved in the interaction between flaviviruses and tick cells and also revealed that there might be antiviral innate immune pathways present in ticks additional to the exogenous RNAi pathway

Influence of water uptake on the aerosol particle light scattering coefficients of the Central European aerosol

The influence of aerosol water uptake on the aerosol particle light scattering was examined at the regional continental research site Melpitz, Germany. The scattering enhancement factor f(RH), defined as the aerosol particle scattering coefficient at a certain relative humidity (RH) divided by its dry value, was measured using a humidified nephelometer. The chemical composition and other microphysical properties were measured in parallel. f(RH) showed a strong variation, e.g. with values between 1.2 and 3.6 at RH=85% and λ=550 nm. The chemical composition was found to be the main factor determining the magnitude of f(RH), since the magnitude of f(RH) clearly correlated with the inorganic mass fraction measured by an aerosol mass spectrometer (AMS). Hysteresis within the recorded humidograms was observed and explained by long-range transported sea salt. A closure study using Mie theory showed the consistency of the measured parameters

Antiviral RNA interference responses induced by Semliki Forest virus infection of mosquito cells: characterization, origin, and frequency-dependent functions of virus-derived small interfering RNAs

RNA interference (RNAi) is an important mosquito defense mechanism against arbovirus infection. In this paper we study the processes underlying antiviral RNAi in Aedes albopictus-derived U4.4 mosquito cells infected with Semliki Forest virus (SFV) (Togaviridae; Alphavirus). The production of virus-derived small interfering RNAs (viRNAs) from viral double-stranded RNA (dsRNA) is a key event in this host response. dsRNA could be formed by RNA replication intermediates, by secondary structures in RNA genomes or antigenomes, or by both. Which of these dsRNAs is the substrate for the generation of viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and bioinformatic analysis of RNA secondary structures to gain insights into the characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived viRNAs with notable areas of high-level viRNA production (hot spots) and no or a low frequency of viRNA production (cold spots) along the length of the viral genome with a slight bias toward the production of genome-derived viRNAs over antigenome-derived viRNAs was observed. Bioinformatic analysis suggests that hot spots of viRNA production are rarely but not generally associated with putative secondary structures in the SFV genome, suggesting that most viRNAs are derived from replicative dsRNA. A pattern of viRNAs almost identical to those of A. albopictus cells was observed for Aedes aegypti-derived Aag2 cells, suggesting common mechanisms that lead to viRNA production. Hot-spot viRNAs were found to be significantly less efficient at mediating antiviral RNAi than cold-spot viRNAs, pointing toward a nucleic acid-based viral decoy mechanism to evade the RNAi response

Expression of Egf1.0 increases mortality of <i>Ae. aegypti</i> and replication of SFV <i>in vivo</i>.

<p>(<b>A</b>) <i>Ae. aegypti</i> were fed blood containing SFV4(3H)-<i>FFLuc</i>-Egf1.0F or SFV4(3H)-<i>FFLuc</i>-Egf1.0R. Uninfected blood meals served as a control. Mosquito mortality was then monitored daily post-bloodmeal. Combined survival data from three independent experiments (cohorts of 22–25 infected mosquitoes per virus or control mosquitoes in each experiment) are shown. Error bars show standard deviation. (<b>B</b>) SFV genome copy number as determined by real time qPCR. Total RNA was extracted 3 days post-bloodmeal from mosquitoes infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F or SFV4(3H)-<i>FFLuc</i>-Egf1.0R. Viral genome RNA levels from 10 mosquitoes for each virus are shown. Values at 0 represent uninfected mosquitoes. Horizontal bar indicates average genome copy number from infected mosquitoes. This experiment was repeated three times with similar results.</p