Bile acid-sensitive human norovirus strains are susceptible to sphingosine-1-phosphate receptor 2 inhibition.

Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.

Widespread nitrous oxide undersaturation in farm waterbodies creates an unexpected greenhouse gas sink.

Nitrogen pollution and global eutrophication are predicted to increase nitrous oxide (N2O) emissions from freshwater ecosystems. Surface waters within agricultural landscapes experience the full impact of these pressures and can contribute substantially to total landscape N2O emissions. However, N2O measurements to date have focused on flowing waters. Small artificial waterbodies remain greatly understudied in the context of agricultural N2O emissions. This study provides a regional analysis of N2O measurements in small (<0.01 km2) artificial reservoirs, of which an estimated 16 million exist globally. We show that 67% of reservoirs were N2O sinks (-12 to -2 µmol N2O⋅m-2⋅d-1) in Canada's largest agricultural area, despite their highly eutrophic status [99 ± 289 µg⋅L-1 chlorophyll-a (Chl-a)]. Generalized additive models indicated that in situ N2O concentrations were strongly and nonlinearly related to stratification strength and dissolved inorganic nitrogen content, with the lowest N2O levels under conditions of strong water column stability and high algal biomass. Predicted fluxes from previously published models based on lakes, reservoirs, and agricultural waters overestimated measured fluxes on average by 7- to 33-fold, challenging the widely held view that eutrophic N-enriched waters are sources of N2O.

Unabated Nitrogen Pollution Favors Growth of Toxic Cyanobacteria over Chlorophytes in Most Hypereutrophic Lakes.

Human release of reactive nitrogen (N) to the environment has increased 10-fold since 1860 and is expected to increase by a further ∼75% by 2050. Much of this N enters phosphorus (P)-rich, eutrophic lakes in agricultural and urbanized watersheds. While N pollution of eutrophic lakes can promote toxic cyanobacterial growth, some cases of extreme N loading have led to the dominance of chlorophytes (green algae). As N loads required to shift communities from cyanobacterial to chlorophyte dominance are unclear, we experimentally tested phytoplankton responses to a gradient of N loading in a P-rich lake. Low-to-moderate doses (1-3 mg N L-1 week-1) promoted toxic cyanobacterial dominance and elevated concentrations of the hepatotoxin microcystin. Conversely, loads characteristic of pure urban or agricultural effluents (up to 18 mg N L-1 week-1) led to the dominance of chlorophytes over cyanobacteria and lower microcystin content. This indicates that N loads needed to sustain chlorophyte dominance are uncommon, likely restricted to select shallow lakes directly exposed to urban or agricultural effluents. As most N pollution regimes in P-rich lakes will favor toxic cyanobacterial dominance, restricting future N pollution will help curb further cyanobacterial dominance in lakes both directly and by constraining the capacity for future P loading and climate warming to drive cyanobacterial growth.

Decoupled trophic responses to long-term recovery from acidification and associated browning in lakes.

Increases in the concentration of dissolved organic matter (DOM) have been documented in many inland waters in recent decades, a process known as "browning". Previous studies have often used space-for-time substitution to examine the direct consequences of increased DOM on lake ecosystems. However, browning often occurs concomitant with other ecologically important water chemistry changes that may interact with or overwhelm any potential ecological response to browning itself. Here we examine a long-term (~20 year) dataset of 28 lakes in the Adirondack Park, New York, USA, that have undergone strong browning in response to recovery from acidification. With these data, we explored how primary producer and zooplankton consumer populations changed during this time and what physical and chemical changes best predicted these long-term ecosystem changes. Our results indicate that changes in primary producers are likely driven by reduced water clarity due to browning, independent of changes in nutrients, counter to previously hypothesized primary producer response to browning. In contrast, declines in calcium concomitant with browning play an important role in driving long-term declines in zooplankton biomass. Our results indicate that responses to browning at different trophic levels are decoupled from one another. Concomitant chemical changes have important implications for our understanding of the response of aquatic ecosystems to browning.

Climate and land use interactively affect lake phytoplankton nutrient limitation status.

Climate-change models predict more frequent and intense summer droughts for many areas, including the midwestern United States. Precipitation quantity and intensity in turn drive the rates and ratios at which nitrogen (N) and phosphorus (P) are exported from watersheds into lakes, but these rates and ratios are also modulated by watershed land use. This led us to ask the question, is the effect of precipitation on phytoplankton nutrient limitation dependent on watershed land use? Across 42 lakes, we found that phytoplankton in lakes in agricultural landscapes were usually P limited but shifted to strong N limitation under increased drought intensity, and that droughts promoted N-fixing cyanobacteria. In contrast, phytoplankton in lakes with forested watersheds were consistently N limited, regardless of drought status. This climate-land use interaction suggests that droughts may increase the incidence of N limitation in agriculturally impacted lakes. N limitation would likely impair valuable ecosystem services such as drinking water, fisheries, and recreation by promoting the occurrence and severity of cyanobacterial blooms.