Optimizing Human Intestinal Enteroids for Environmental Monitoring of Human Norovirus

Human noroviruses (HuNoV) are the leading cause of gastrointestinal illness and environmental monitoring is crucial to prevent HuNoV outbreaks. The recent development of a HuNoV cell culture assay in human intestinal enteroids (HIEs) has enabled detection of infectious HuNoV. However, this complex approach requires adaptation of HIEs to facilitate HuNoV replication from environmental matrixes. Integrating data from 200 experiments, we examined six variables: HIE age, HIE basement membrane compounds (BMC), HuNoV inoculum processing, HuNoV inoculum volume, treatment of data below limit of detection (LOD), and cutoff criteria for determining positive HuNoV growth. We infected HIEs with HuNoV GII.4 Sydney positive stool and determined 1.4 × 10³ genome equivalents per HIE well were required for HuNoV replication. HIE age had minimal effect on assay outcomes. LOD replacement and cutoff affected data interpretation, with lower values resulting in higher estimated HuNoV detection. Higher inoculum volumes lead to minimal decreases in HuNoV growth, with an optimal volume of 250uL facilitating capture of low concentrations of HuNoVs present in environmental isolates. Processing of HuNoV inoculum is valuable for disinfection studies and concentrating samples but is not necessary for all HIE applications. This work enhances the HuNoV HIE cell culture approach for environmental monitoring. Future HIE research should report cell age as days of growth and should clearly describe BMC choice, LOD handling, and positive cutoff.

     

Better the nest site you know: decision-making during nest migrations by the Pharaoh’s ant

Animals frequently have to decide between alternative resources and in social insects these individual choices produce a colony-level decision. The choice of nest site is a particularly critical decision for a social insect colony to make, but the decision making process has still only been studied in a few species. In this study, we investigated nest selection by the Pharaoh’s ant, Monomorium pharaonis, a species renowned for its propensity to migrate and its use of multi-component trail pheromones to organise decision-making in other contexts. When presented with the choice of familiar and novel nests of equal quality in a Y set-up, colonies preferentially migrated towards the familiar nest, suggesting a form of colony-level ‘memory’ of potential nest sites. However, if the novel nest was superior to the familiar nest, then colonies began migrating initially to the familiar nest, but then redirected their migration to the superior quality novel nest. This may be an effective method of reducing colony exposure while searching for an optimum nest site. Branches that had previously led to a selected nest were attractive to ants in subsequent migrations, suggesting that trail pheromones mediate the decision making process. The adaptive, pheromone-based organisation of nest-site selection by Pharaoh’s ants matches their ephemeral environment and is likely to contribute to their success as a 'tramp' species.     

Mating opportunities,paternity, and sexual conflict: paternal care in northern and southern temperate house wrens

Males are generally predicted to care less for their young when they have more additional mating opportunities, lower paternity, or when their mates care more. We tested these predictions using male provisioning as a proxy for paternal care in two temperate populations of house wrens (Troglodytes aedon) with divergent life histories. Males in the migratory, occasionally socially polygynous New York, USA (northern) population provisioned less when more local females were fertile. A similar relationship was only weakly supported in the resident, socially monogamous Buenos Aires Province, Argentina (southern) population, possibly due to the higher density of house wrens there. A relationship between male provisioning and level of paternity within the brood was supported in both populations, but in opposite directions: while males in the southern population provisioned less at broods containing more extra-pair young, males in the northern population provisioned such broods more, contradicting predictions. Males provisioned less when their mates provisioned more in both populations, in agreement with sexual conflict theory. Additionally, the populations both exhibited a positive relationship between male provisioning and nestling age, but differed in the direction of the relationships of male provisioning with date and brood size. Our results suggest that even within a species, life history differences may be accompanied by differences in the determinants of behavior such as paternal care.     

Sediment load and timing of sedimentation affect spore establishment in Macrocystis pyrifera and Undaria pinnatifida

Although the frequency and magnitude of sedimentation often varies across coastal landscapes creating patches with different mean sediment loads, duration of sedimentation and rates of sediment resuspension, few studies have documented the emergent effects of spatio-temporal variability in sedimentation. Here, we conducted two laboratory experiments to evaluate such effects on the establishment of Macrocystis pyrifera and Undaria pinnatifida spores. In the first experiment, spore establishment was significantly affected by sediment load (the effective dose required for a 40 % reduction in establishment ranged between 16 and 60 mg sediment l^?1) and sediment regime (relative sedimentation occurring before spore settlement, ~3 times more sediment was required for 20 % reduction in spore establishment when sedimentation occurred after spore settlement). The second experiment demonstrated that the effects of sediment depended on sediment load (spore establishment was 2–4 times greater when sediment load was 200 mg l^?1 relative to 400 mg l^?1), variability in sedimentation (spore establishment was 1.36 times greater with variable than fixed sediment loads), repeated pulses of sedimentation (pulsed sedimentation decreased spore establishment by 59–91 % relative to a single sedimentation event) and timing of sedimentation relative to spore settlement (sedimentation before spore settlement decreased establishment by 51–95 % relative to sedimentation after spore settlement). These results have important implications for ecologists and resource managers attempting to predict the consequences of sedimentation, suggesting that it is not only important to consider sediment load, but also fine-scale temporal variability in sedimentation relative to key life-history events of the impacted organisms.     

Threshold temperatures mediate the impact of reduced snow cover on overwintering freeze-tolerant caterpillars

Decreases in snow cover due to climate change could alter the energetics and physiology of ectothermic animals that overwinter beneath snow, yet how snow cover interacts with physiological thresholds is unknown. We applied numerical simulation of overwintering metabolic rates coupled with field validation to determine the importance of snow cover and freezing to the overwintering lipid consumption of the freeze-tolerant Arctiid caterpillar Pyrrharctia isabella. Caterpillars that overwintered above the snow experienced mean temperatures 1.3°C lower than those below snow and consumed 18.36 mg less lipid of a total 68.97-mg reserve. Simulations showed that linear temperature effects on metabolic rate accounted for only 30% of the difference in lipid consumption. When metabolic suppression by freezing was included, 93% of the difference between animals that overwintered above and below snow was explained. Our results were robust to differences in temperature sensitivity of metabolic rate, changes in freezing point, and the magnitude of metabolic suppression by freezing. The majority of the energy savings was caused by the non-continuous reduction in metabolic rate due to freezing, the first example of the importance of temperature thresholds in the lipid use of overwintering insects.     

Biomass rather than growth rate determines variation in net primary production by giant kelp

Net primary production (NPP) is influenced by disturbance-driven fluctuations in foliar standing crop (FSC) and resource-driven fluctuations in rates of recruitment and growth, yet most studies of NPP have focused primarily on factors influencing growth. We quantified NPP, FSC, recruitment, and growth rate for the giant kelp, Macrocystis pyrifera, at three kelp forests in southern California, U.S.A., over a 54-month period and determined the relative roles of FSC, recruitment, and growth rate in contributing to variation in annual NPP. Net primary production averaged between 0.42 and 2.38 kg dry mass x m(-2) x yr(-1) at the three sites. The initial FSC present at the beginning of the growth year and the recruitment of new plants during the year explained 63% and 21% of the interannual variation observed in NPP, respectively. The previous year's NPP and disturbance from waves collectively accounted for 80% of the interannual variation in initial FSC. No correlation was found between annual growth rate (i.e., the amount of new kelp mass produced per unit of existing kelp mass) and annual NPP (i.e., the amount of new kelp mass produced per unit area of ocean bottom), largely because annual growth rate was consistent compared to initial FSC and recruitment, which fluctuated greatly among years and sites. Although growth rate was a poor predictor of variation in annual NPP, it was principally responsible for the high mean values observed for NPP by Macrocystis. These high mean values reflected rapid growth (average of approximately 2% per day) of a relatively small standing crop (maximum annual mean = 444 g dry mass/m2) that replaced itself approximately seven times per year. Disturbance-driven variability in FSC may be generally important in explaining variation in NPP, yet it is rarely examined because cycles of disturbance and recovery occur over timescales of decades or more in many systems. Considerable insight into how variation in FSC drives variation in NPP may be gained by studying systems such as giant kelp forests that are characterized by frequent disturbance and rapid rates of growth and recruitment.     

Initial effects of light armored vehicle use on grassland vegetation at Fort Lewis, Washington

Sustainable use of military training lands requires understanding and prediction of the effects of military vehicles on vegetation. We examined the initial impacts of an 8-wheeled, light armored vehicle (LAV) on grassland vegetation at Fort Lewis, Washington. The LAV drove replicate spiral paths at two starting velocities, 10.3 and 5.1 m s(-1). The disturbed width (width of ground impacted by the tires) increased as turning radius decreased, but was unaffected by vehicle velocity. An inverse-exponential model predicted disturbed width (r(2)=0.68) at all turning radii for both velocities combined. In low-velocity spirals, and for straight tracking (turning radius>40 m) and moderate turns (radius 20-40 m) in high-velocity spirals, all vegetation damage was imprint (plants flattened by wheels). During sharp (radius <20 m), high-velocity turns, most or all of the disturbed width was scraped free of surface vegetation and soil, which was piled to the outside of each tire track. Total plant cover (all species) was not affected by track curvature in low-velocity spirals, but decreased in the order straight tracking>moderate turns>sharp turns in high-velocity spirals. In low-velocity spirals, post-tracking cover of several plant growth forms (non-native species, perennial species, sod-forming grasses) was similar to pre-tracking cover, but in high-velocity spirals, post-tracking cover of these growth forms decreased in the order straight > or =moderate=sharp. Cover of native species and forbs decreased more in high- than in low-velocity spirals, but was unaffected by curvature. Pre- and post-tracking cover of annual species, bunchgrasses, and shrubs was < or =3%. The most severe vegetation damage caused by operation of wheeled LAVs on grasslands is associated with sharp, high-velocity turns.     

Impacts of pesticides in a Central California estuary

Recent and past studies have documented the prevalence of pyrethroid and organophosphate pesticides in urban and agricultural watersheds in California. While toxic concentrations of these pesticides have been found in freshwater systems, there has been little research into their impacts in marine receiving waters. Our study investigated pesticide impacts in the Santa Maria River estuary, which provides critical habitat to numerous aquatic, terrestrial, and avian species on the central California coast. Runoff from irrigated agriculture constitutes a significant portion of Santa Maria River flow during most of the year, and a number of studies have documented pesticide occurrence and biological impacts in this watershed. Our study extended into the Santa Maria watershed coastal zone and measured pesticide concentrations throughout the estuary, including the water column and sediments. Biological effects were measured at the organism and community levels. Results of this study suggest the Santa Maria River estuary is impacted by current-use pesticides. The majority of water samples were highly toxic to invertebrates (Ceriodaphnia dubia and Hyalella azteca), and chemistry evidence suggests toxicity was associated with the organophosphate pesticide chlorpyrifos, pyrethroid pesticides, or mixtures of both classes of pesticides. A high percentage of sediment samples were also toxic in this estuary, and sediment toxicity occurred when mixtures of chlorpyrifos and pyrethroid pesticides exceeded established toxicity thresholds. Based on a Relative Benthic Index, Santa Maria estuary stations where benthic macroinvertebrate communities were assessed were degraded. Impacts in the Santa Maria River estuary were likely due to the proximity of this system to Orcutt Creek, the tributary which accounts for most of the flow to the lower Santa Maria River. Water and sediment samples from Orcutt Creek were highly toxic to invertebrates due to mixtures of the same pesticides measured in the estuary. This study suggests that the same pyrethroid and organophosphate pesticides that have been shown to cause water and sediment toxicity in urban and agriculture water bodies throughout California, have the potential to affect estuarine habitats. The results establish baseline data in the Santa Maria River estuary to allow evaluation of ecosystem improvement as management initiatives to reduce pesticide runoff are implemented in this watershed.     

The acoustic structure of chimpanzee pant-hooting facilitates chorusing

Duetting or chorusing behaviour occurs in a wide variety of animals and is posited to fulfil various important functions including territory defence and social bonding. The structure of calls produced in choruses might be shaped in a way that facilitates such joint vocal displays. In this study, we test the hypothesis that flexibility to modify the temporal structure of chimpanzee pant-hoots, vocalisations often given jointly with other individuals, facilitates chorusing. The results of this study, which was conducted on two communities of wild chimpanzees in Uganda, support this hypothesis. First, the duration of the build-up phase of the pant-hoot correlated with the latency with which the partner joined in the call, suggesting that males prolong the duration of the build-up to allow others to join in the call and to increase the likelihood of a chorus occurring. Second, the loud climax phases were significantly longer when produced in choruses than alone, which suggests that males prolong this part of the call when calling in choruses. Within chorus pant-hoots, there was a positive relationship between the number of climax elements given by two calling partners, suggesting that males adjust the temporal structure of their call to mirror their partner's call. We conclude that the basic acoustic structure of chimpanzee pant-hoots and the flexibility with which males adjust the duration of the constituent phases promote chorusing, and that the temporal structure of this rather stereotyped vocalisation is sensitive to fine details of the vocal responses of the audience.