A high-throughput technique to map cell images to cell positions using a 3D imaging flow cytometer.

We develop a high-throughput technique to relate positions of individual cells to their three-dimensional (3D) imaging features with single-cell resolution. The technique is particularly suitable for nonadherent cells where existing spatial biology methodologies relating cell properties to their positions in a solid tissue do not apply. Our design consists of two parts, as follows: recording 3D cell images at high throughput (500 to 1,000 cells/s) using a custom 3D imaging flow cytometer (3D-IFC) and dispensing cells in a first-in-first-out (FIFO) manner using a robotic cell placement platform (CPP). To prevent errors due to violations of the FIFO principle, we invented a method that uses marker beads and DNA sequencing software to detect errors. Experiments with human cancer cell lines demonstrate the feasibility of mapping 3D side scattering and fluorescent images, as well as two-dimensional (2D) transmission images of cells to their locations on the membrane filter for around 100,000 cells in less than 10 min. While the current work uses our specially designed 3D imaging flow cytometer to produce 3D cell images, our methodology can support other imaging modalities. The technology and method form a bridge between single-cell image analysis and single-cell molecular analysis.

Soil Arbuscular Mycorrhizal Fungal Communities Differentially Affect Growth and Nutrient Uptake by Grapevine Rootstocks.

Arbuscular mycorrhizal fungi (AMF) deliver potentially significant services in sustainable agricultural ecosystems, yet we still lack evidence showing how AMF abundance and/or community composition can benefit crops. In this study, we manipulated AMF communities in grapevine rootstock and measured plant growth and physiological responses. Glasshouse experiments were set up to determine the interaction between rootstock variety and different AMF communities, using AMF communities originating under their own (i.e., "home") soil and other rootstocks' (i.e., "away") soil. The results revealed that specific AMF communities had differential effects on grapevine rootstock growth and nutrient uptake. It was demonstrated that a rootstock generally performed better in the presence of its own AMF community. This study also showed that AMF spore diversity and the relative abundance of certain species is an important factor as, when present in equal abundance, competition between species was indicated to occur, resulting in a reduction in the positive growth outcomes. Moreover, there was a significant difference between the communities with some AMF communities increasing plant growth and nutrient uptake compared with others. The outcomes also demonstrated that some AMF communities indirectly influenced the chlorophyll content in grapevine leaves through the increase of specific nutrients such as K, Mn, and Zn. The findings also indicated that some AMF species may deliver particular benefits to grapevine plants. This work has provided an improved understanding of community level AMF-grapevine interaction and delivered an increased knowledge of the ecosystem services they provide which will benefit the wine growers and the viticulture industry.

Nest-type associated microclimatic conditions as potential drivers of ectoparasite infestations in African penguin nests.

Nest design and characteristics can influence the microclimatic conditions in the nest. Nest-dwelling ectoparasites are sensitive to temperature and moisture and as such the conditions in the nest can influence parasite infestations. The endangered African penguin (Spheniscus demersus) breeds in different nest types and as yet little is known with regard to the microclimate and parasite infestation within these nests. This study characterized the microclimatic conditions in natural open, natural covered (with vegetation) and artificial nests, and assessed the relationship between nest characteristics (type, age, distance from the coast, orientation and entrance opening) and in-nest ectoparasite infestations and the health of African penguins in Stony Point, South Africa. Penguins (50 adults and 192 chicks) and their nests (n = 308) were sampled in 2016 and 2017. Soil temperature was higher in artificial than in natural nests, and soil and nest material moisture was lower in artificial and natural covered nests than natural open. Ectoparasite infestations were higher under warmer and drier conditions, in artificial nests and nests near the coastline. Penguin (adult and chick) body mass and chick body condition were lower in warmer nests and total plasma protein (in adults and checks) was lower in drier nests. Given the potential adverse effects of ectoparasites on host species, it is recommended that conservation agencies implement a monitoring programme to assess the ectoparasite infestation in artificial nests across multiple colonies. This information will facilitate a more holistic penguin conservation management plan that may prevent further detrimental effects on this endangered penguin species.

Parasite diversity associated with African penguins (Spheniscus demersus) and the effect of host and environmental factors.

The African penguin (Spheniscus demersus) is a critically endangered species endemic to southern Africa. Limited information is available on the parasite diversity associated with the species in natural settings. This study explores the diversity and incidence of parasites associated with African penguins and their nests, and records the effect of host and environmental factors on parasite infestation. Ecto-, haemo- and helminth parasites were recorded from 210 adult birds, 583 chicks and 628 nests across five colonies (two mainland and three islands) along the south-western coast of South Africa, in 2016 and 2017. Mean nest density (total and active nests) and climate variables (temperature and precipitation) were obtained for each colony. Parapsyllus humboldti was the most abundant and prevalent ectoparasite on penguins and in nests (69.10 and 57.80%, respectively), while Piroplasmorida/Haemospororida (33.51%) and Cardiocephaloides spp. (56.17%) were the most prevalent haemo- and helminth parasites of penguins, respectively. In general parasite abundance and prevalence was significantly affected by penguin age (chicks vs adults), location (mainland vs islands), nest density (total and active nests) and season (spring vs autumn/winter). It is concluded that parasite infestations are structured and that penguin chicks at mainland colonies are more susceptible to parasite infestations during spring.

Trait differences in responses to arbuscular mycorrhizal fungi are stronger and more consistent than fixed differences among populations of Asclepias speciosa.

PREMISE OF THE STUDY: Arbuscular mycorrhizal (AM) fungi can promote plant growth and reproduction, but other plant physiological traits or traits that provide defense against herbivores can also be affected by AM fungi. However, whether responses of different traits to AM fungi are correlated and whether these relationships vary among plants from different populations are unresolved. METHODS: In a common garden experiment, we grew Asclepias speciosa plants from seed collected from populations found along an environmental gradient with and without AM fungi to assess whether the responses of six growth and defense traits to AM fungi are correlated. KEY RESULTS: Although there was strong genetic differentiation in mean trait values among populations, AM fungi consistently increased expression of most growth and defense traits across all populations. Responses of biomass and root to shoot ratio to AM fungi were positively correlated, suggesting that plants that are more responsive to AM fungi allocated more biomass belowground. Responses of biomass and trichome density to AM fungi were negatively correlated, indicating a trade-off in responsiveness between a growth and defensive trait. CONCLUSIONS: Our results suggest that while there is substantial population differentiation in many traits of A. speciosa, populations respond similarly to AM fungi, and both positive and negative correlations among trait responses occur.

The emerging science of linked plant-fungal invasions.

Contents 1314 I. 1315 II. 1316 III. 1322 IV. 1323 V. 1325 VI. 1326 VII. 1326 VIII. 1327 1328 References 1328 SUMMARY: Invasions of alien plants are typically studied as invasions of individual species, yet interactions between plants and symbiotic fungi (mutualists and potential pathogens) affect plant survival, physiological traits, and reproduction and hence invasion success. Studies show that plant-fungal associations are frequently key drivers of plant invasion success and impact, but clear conceptual frameworks and integration across studies are needed to move beyond a series of case studies towards a more predictive understanding. Here, we consider linked plant-fungal invasions from the perspective of plant and fungal origin, simplified to the least complex representations or 'motifs'. By characterizing these interaction motifs, parallels in invasion processes between pathogen and mutualist fungi become clear, although the outcomes are often opposite in effect. These interaction motifs provide hypotheses for fungal-driven dynamics behind observed plant invasion trajectories. In some situations, the effects of plant-fungal interactions are inconsistent or negligible. Variability in when and where different interaction motifs matter may be driven by specificity in the plant-fungal interaction, the size of the effect of the symbiosis (negative to positive) on plants and the dependence (obligate to facultative) of the plant-fungal interaction. Linked plant-fungal invasions can transform communities and ecosystem function, with potential for persistent legacies preventing ecosystem restoration.

Cascading effects of fire retardant on plant-microbe interactions, community composition, and invasion.

Climate change, historical fire suppression, and a rise in human movements in urban-forest boundaries have resulted in an increased use of long-term fire retardant (LTFR). While LTFR is an effective fire-fighting tool, it contains high concentrations of nitrogen and phosphorus, and little is known about how this nutrient pulse affects terrestrial ecosystems. We used field surveys and greenhouse experiments to quantify effects of LTFR on plant productivity, community composition, and plant interactions with the ubiquitous root symbiont arbuscular mycorrhizal fungi (AMF). In the field, LTFR applications were associated with persistent shifts in plant communities toward exotic annuals with little or no dependency of AMF. Plants exposed to LTFR were less colonized by AMF, both in field surveys and in the greenhouse, and this was most likely due to the substantial and persistent increase in soil available phosphorus. All plants grew bigger with LTFR in the greenhouse, but the invasive annual cheatgrass (Bromus tectorum) benefitted most. While LTFR can control fires, it may cause long-term changes in soil nutrient availabilities, disrupt plant interactions with beneficial soil microbes, and exasperate invasion by some exotic plants.

Navigating the labyrinth: a guide to sequence-based, community ecology of arbuscular mycorrhizal fungi.

Data generated from next generation sequencing (NGS) will soon comprise the majority of information about arbuscular mycorrhizal fungal (AMF) communities. Although these approaches give deeper insight, analysing NGS data involves decisions that can significantly affect results and conclusions. This is particularly true for AMF community studies, because much remains to be known about their basic biology and genetics. During a workshop in 2013, representatives from seven research groups using NGS for AMF community ecology gathered to discuss common challenges and directions for future research. Our goal was to improve the quality and accessibility of NGS data for the AMF research community. Discussions spanned sampling design, sample preservation, sequencing, bioinformatics and data archiving. With concrete examples we demonstrated how different approaches can significantly alter analysis outcomes. Failure to consider the consequences of these decisions may compound bias introduced at each step along the workflow. The products of these discussions have been summarized in this paper in order to serve as a guide for any researcher undertaking NGS sequencing of AMF communities.

Puke or poop? Comparison of regurgitate and faecal samples to infer alpine grasshopper (Paprides nitidus Hutton) diet in experimental plant communities.

Characterising plant-herbivore interactions is important to understanding the processes that influence community structure and ecosystem functioning. Traditional methods used to identify plant-herbivore interactions are being superseded by non-destructive molecular approaches that can infer interactions with greater resolution and accuracy from environmental DNA (e.g. faeces and regurgitate). However, few studies have compared the success of using different sample types and whether they provide similar or contrasting information about species' diet. Here we compared the success of DNA amplification and host plant species identification using restriction fragment length polymorphism (RFLP) applied to faecal and regurgitate samples collected from alpine grasshoppers Paprides nitidus Hutton during a grassland community mesocosm experiment. We found that DNA amplification success was 23% and 86% higher for faecal than regurgitate samples from female and male grasshoppers, respectively. In contrast, successful host plant identification using RFLP was 9% higher for regurgitate than faecal samples. The mean number of host plant species identified per sample (1.40) did not differ between sample types or grasshopper sexes. Of the 136 paired faecal-regurgitate samples, just 41% and 74% produced exactly or partially matching host plant identifications, respectively, indicating that different sample types provided complementary information about herbivore diet. Some plant species were more likely to be identified from faecal samples than expected by chance, and we found that this identification bias skewed towards plant species with higher investment in leaf tissue. We conclude that multiple sample types may be required to fully characterise an invertebrate herbivore species' diet.

Factors determining the number of seabirds impacted by oil spills and the success of their rehabilitation: Lessons learned from Namibia and South Africa.

The coastal waters of Namibia and South Africa have an extensive history of oil spills, with 71 recorded up to 2021. Thirty-nine spills reportedly affected 83,224 seabirds, with African penguins (Spheniscus demersus; 91.0 %) and Cape gannets (Morus capensis; 8.5 %) most affected. Spills affecting seabirds were caused by unknown sources (46 %), bulk/cargo carriers (43 %), tankers (38 %) and ship-to-ship transfers (14 %). The number of penguins oiled was predicted by the breeding population size within 25 to 75 km, but not the volume of oiled spilled, the month or the year. Rehabilitation records from penguins oiled in spills since 2001 reveal that the day of admission (relative to the start of the spill) was predictive of packed cell volume, body mass, and plasma total solids, with the latter two being predictive of rehabilitation success. Our results highlight the importance of rapid monitoring at colonies to locate oiled birds in the event of spills.

Plant species identity and plant-induced changes in soil physicochemistry-but not plant phylogeny or functional traits - shape the assembly of the root-associated soil microbiome.

The root-associated soil microbiome contributes immensely to support plant health and performance against abiotic and biotic stressors. Understanding the processes that shape microbial assembly in root-associated soils is of interest in microbial ecology and plant health research. In this study, 37 plant species were grown in the same soil mixture for 10 months, whereupon the root-associated soil microbiome was assessed using amplicon sequencing. From this, the contribution of direct and indirect plant effects on microbial assembly was assessed. Plant species and plant-induced changes in soil physicochemistry were the most significant factors that accounted for bacterial and fungal community variation. Considering that all plants were grown in the same starting soil mixture, our results suggest that plants, in part, shape the assembly of their root-associated soil microbiome via their effects on soil physicochemistry. With the increase in phylogenetic ranking from plant species to class, we observed declines in the degree of community variation attributed to phylogenetic origin. That is, plant-microbe associations were unique to each plant species, but the phylogenetic associations between plant species were not important. We observed a large degree of residual variation (> 65%) not accounted for by any plant-related factors, which may be attributed to random community assembly.

Low-latency label-free image-activated cell sorting using fast deep learning and AI inferencing.

Classification and sorting of cells using image-activated cell sorting (IACS) systems can bring significant insight to biomedical sciences. Incorporating deep learning algorithms into IACS enables cell classification and isolation based on complex and human-vision uninterpretable morphological features within a heterogeneous cell population. However, the limited capabilities and complicated implementation of deep learning-assisted IACS systems reported to date hinder the adoption of the systems for a wide range of biomedical research. Here, we present image-activated cell sorting by applying fast deep learning algorithms to conduct cell sorting without labeling. The overall sorting latency, including signal processing and AI inferencing, is less than 3 ms, and the training time for the deep learning model is less than 30 min with a training dataset of 20,000 images. Both values set the record for IACS with sorting by AI inference. . We demonstrated our system performance through a 2-part polystyrene beads sorting experiment with 96.6% sorting purity, and a 3-part human leukocytes sorting experiment with 89.05% sorting purity for monocytes, 92.00% sorting purity for lymphocytes, and 98.24% sorting purity for granulocytes. The above performance was achieved with simple hardware containing only 1 FPGA, 1 PC and GPU, as a result of an optimized custom CNN UNet and efficient use of computing power. The system provides a compact, sterile, low-cost, label-free, and low-latency cell sorting solution based on real-time AI inferencing and fast training of the deep learning model.

Population-level compensation impedes biological control of an invasive forb and indirect release of a native grass.

The intentional introduction of specialist insect herbivores for biological control of exotic weeds provides ideal but understudied systems for evaluating important ecological concepts related to top-down control, plant compensatory responses, indirect effects, and the influence of environmental context on these processes. Centaurea stoebe (spotted knapweed) is a notorious rangeland weed that exhibited regional declines in the early 2000s, attributed to drought by some and to successful biocontrol by others. We initiated an experiment to quantify the effects of the biocontrol agent, Cyphocleonus achates, on Ce. stoebe and its interaction with a dominant native grass competitor, Pseudoroegneria spicata, under contrasting precipitation conditions. Plots containing monocultures of each plant species or equal mixtures of the two received factorial combinations of Cy. achates herbivory (exclusion or addition) and precipitation (May-June drought or "normal," defined by the 50-year average) for three years. Cy. achates herbivory reduced survival of adult Ce. stoebe plants by 9% overall, but this effect was stronger under normal precipitation compared to drought conditions, and stronger in mixed-species plots compared to monocultures. Herbivory had no effect on Ce. stoebe per capita seed production or on recruitment of seedlings or juveniles. In normal-precipitation plots of mixed composition, greater adult mortality due to Cy. achates herbivory resulted in increased recruitment of new adult Ce. stoebe. Due to this compensatory response to adult mortality, final Ce. stoebe densities did not differ between herbivory treatments regardless of context. Experimental drought reduced adult Ce. stoebe survival in mixed-species plots but did not impede recruitment of new adults or reduce final Ce. stoebe densities, perhaps due to the limited duration of the treatment. Ce. stoebe strongly depressed P. spicata reproduction and recruitment, but these impacts were not substantively alleviated by herbivory on Ce. stoebe. Population-level compensation by dominant plants may be an important factor inhibiting top-down effects in herbivore-driven and predator-driven cascades.

Factors that influence Cape fur seal predation on Cape gannets at Lambert's Bay, South Africa.

Seabird populations experience predation that can impact their breeding density and breeding success. The Cape gannet Morus capensis is endemic to the Benguela upwelling ecosystem and is classified as Endangered by the IUCN. They are affected by several threats, including predation by the Cape fur seal Arctocephalus pusillus pusillus. Many fledglings succumb to predation during their maiden flight across waters around the island. To curb predation, the selective culling of individual predatory seals was implemented in 2014, 2015, and 2018. Our first study objective was to determine if selective culling of Cape fur seals significantly reduced predation probability on Cape gannets. We tested whether predation probability in 2014, 2015, and 2018 was affected by fish biomass, gannet fledgling numbers, and/or the presence/absence of selective culling. Our second objective was to determine what led to fluctuations in Cape fur seal predation on Cape gannet fledglings between 2007 and 2018. We tested whether fish biomass and the amount of Cape gannet fledglings in the water affected predation probability on the fledglings. Results indicated that selective culling reduced predation within years. We found that with both increased fledgling numbers and increased fish biomass, seal predation probability was reduced. This suggests that a sustainable way to promote the conservation of Cape gannets would be to increase food availability for both the Cape fur seals and Cape gannets. Our findings, collectively with the global trend of the declining Cape gannet population and their endemism, provide reasons advocating for the conservation of the food resources of both the Cape fur seal and the Cape gannet in the Benguela system.

High Sensitivity, Rapid Detection of Virus in High Traffic Environments.

The global pandemic caused by the SARS-CoV-2 virus has underscored the need for rapid, simple, scalable, and high-throughput multiplex diagnostics in non-laboratory settings. Here we demonstrate a multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a gold nanoparticle-based lateral flow immunoassay (LFIA) capable of detecting up to three unique viral gene targets in 15 min. RT-LAMP primers associated with three separate gene targets from the SARS-CoV-2 virus (Orf1ab, Envelope, and Nucleocapsid) were added to a one-pot mix. A colorimetric change from red to yellow occurs in the presence of a positive sample. Positive samples are run through a LFIA to achieve specificity on a multiplex three-test line paper assay. Positive results are indicated by a characteristic crimson line. The device is almost fully automated and is deployable in any community setting with a power source.

Intercolony variation in reproductive skipping in the African penguin.

In long-lived species, reproductive skipping is a common strategy whereby sexually mature animals skip a breeding season, potentially reducing population growth. This may be an adaptive decision to protect survival, or a non-adaptive decision driven by individual-specific constraints. Understanding the presence and drivers of reproductive skipping behavior can be important for effective population management, yet in many species such as the endangered African penguin (Spheniscus demersus), these factors remain unknown. This study uses multistate mark-recapture methods to estimate African penguin survival and breeding probabilities at two colonies between 2013 and 2020. Overall, survival (mean ± SE) was higher at Stony Point (0.82 ± 0.01) than at Robben Island (0.77 ± 0.02). Inter-colony differences were linked to food availability; under decreasing sardine (Sardinops sagax) abundance, survival decreased at Robben Island and increased at Stony Point. Additionally, reproductive skipping was evident across both colonies; at Robben Island the probability of a breeder becoming a nonbreeder was ~0.22, versus ~0.1 at Stony Point. Penguins skipping reproduction had a lower probability of future breeding than breeding individuals; this lack of adaptive benefit suggests reproductive skipping is driven by individual-specific constraints. Lower survival and breeding propensity at Robben Island places this colony in greater need of conservation action. However, further research on the drivers of inter-colony differences is needed.

A network perspective for sustainable agroecosystems.

Nature-based management aims to improve sustainable agroecosystem production, but its efficacy has been variable. We argue that nature-based agroecosystem management could be significantly improved by explicitly considering and manipulating the underlying networks of species interactions. A network perspective can link species interactions to ecosystem functioning and stability, identify influential species and interactions, and suggest optimal management approaches. Recent advances in predicting the network roles of species from their functional traits could allow direct manipulation of network architecture through additions or removals of species with targeted traits. Combined with improved understanding of the structure and dynamics of networks across spatial and temporal scales and interaction types, including social-ecological, applying these tools to nature-based management can contribute to sustainable agroecosystems.

Escape from competition: neighbors reduce Centaurea stoebe performance at home but not away.

The greater abundance of some exotic plants in their nonnative ranges might be explained in part by biogeographic differences in the strength of competition, but these competitive effects have not been experimentally examined in the field. We compared the effects of neighbors on the growth and reproduction of spotted knapweed (Centaurea stoebe) in Europe, where it is native, and in Montana, where it is invasive. There were strong negative competitive effects of neighboring vegetation on C. stoebe growth and reproduction in Europe. In contrast, identical experiments in Montana resulted in insignificant impacts on C. stoebe. Although the mechanisms that produce this dramatic biogeographic difference in competitive outcome remain unknown, our results indicate that differences in net competitive interactions between ranges may contribute to the striking dominance of C. stoebe in parts of North America.