RootScan: Software for high-throughput analysis of root anatomical traits

Background and aims

RootScan is a program for semi-automated image analysis of anatomical traits in root cross-sections.

Methods

RootScan uses pixel thresholds to separate the cross-section from its background and to divide it into tissue regions. Area measurements and object counts are performed within various regions of interest. A graphical user interface permits the user to see which regions are selected, to edit those selections, and to rate and comment on the data. The structure of the program allows for organized workflow and increased data collection efficiency.

Results

The program collects data on more than 20 variables per image including areas of the cross-section, stele, cortex, aerenchyma lacunae, xylem vessels, and counts of cortical cells and cell files. An increased rate of data collection allows collection of four times more variables in less time than is possible with current methods. Correlation analysis shows that RootScan data is equal or greater in accuracy than data collected with Photoshop.

Conclusions

Compared with currently available tools, this software offers considerable improvements in the amount and quality of data, ease of use, and time needed for data collection. RootScan permits phenotypic scoring of physiologically and agronomically important traits on a large number of genotypes.     

Optimizing Diversity Assessment Protocols for High Canopy Ants in Tropical Rain Forest

The canopies of tropical rain forests support highly diverse, yet poorly known, animal and plant communities. It is vital that researchers who invest the time needed to gain access to the high canopy are able efficiently to survey the animals and plants that they find there. Here, we develop diversity assessment protocols for one of the most ecologically important canopy animal groups, the ants, in lowland dipterocarp rain forest in Sabah, Malaysia. We design and test a novel trap (the purse‐string trap) that can be remotely collected, thus avoiding disturbance to ants. We compare this modified trap with two other methods for surveying canopy ants: precision insecticide fogging and baited pitfall trapping. In total, we collected 39,351 ants belonging to 173 species in 38 genera. Fogging collected the most individuals and species, followed by purse‐string trapping with baited pitfall trapping catching the fewest. Fogging also resulted in samples with a different species composition to purse‐string trapping and baited pitfall trapping, which were not different from one another. Using a ‘greedy algorithm’, which guides the selection of inventory methods in order to maximize new species discovered per researcher‐hour, we show that projects allocating fewer than 132 researcher‐hours to canopy ant collection and identification should sample exclusively using fogging. Those with more time should use a combination of methods. This prioritization technique could be used to accelerate species discovery in future rapid biodiversity assessments. Abstract in Malay is available in the online version of this article.     

Hydrated mucilage reduces post-dispersal seed removal of a sand desert shrub by ants in a semiarid ecosystem

Post-dispersal seed removal by animals can lead to extensive seed loss and thus is an important factor in structuring plant communities. However, we know much less about post-dispersal seed predation than about other forms of herbivory. Mucilage plays many ecological roles in adaptation of plants to diverse environments; nevertheless, until now the role of mucilage in ant-mediated seed movement remains largely hypothetical. We studied the role of mucilage in seed removal of Artemisia sphaerocephala by ants in Mu Us Sandland in Inner Mongolia, China. Messor aciculatus was the most active seed predator of Artemisia sphaerocephala. Time to first ant collecting (T _1st) of wet intact seeds was longest and significantly different from that for dry intact seeds, wet demucilaged seeds, and dry demucilaged seeds; number of seeds removed to ant nests was lowest for wet intact seeds. After they were collected by ants, 5 % of wet intact seeds were dropped during transport. Our results indicate that seed mucilage of Artemisia sphaerocephala may play a significant role in post-dispersal seed removal by (1) making seeds less attractive to ants, thus resulting in a delay of collection time; (2) forming a strong bond to soil particles, making it difficult for ants to remove seeds; and (3) making seeds more likely to be dropped during transport, thereby allowing them to escape from predation even after collection by ants. This study demonstrates the importance of mucilage in reducing seed removal by ants and thus in anchoring seeds of desert plants in the vicinity of mother plants.     

The importance of within-year repeated counts and the influence of scale on long-term monitoring of sage-grouse

Long-term population monitoring is the cornerstone of animal conservation and management. The accuracy and precision of models developed using monitoring data can be influenced by the protocols guiding data collection. The greater sage-grouse (Centrocercus urophasianus) is a species of concern that has been monitored over decades, primarily, by counting the number of males that attend lek (breeding) sites. These lek count data have been used to assess long-term population trends and for multiple mechanistic studies. However, some studies have questioned the efficacy of lek counts to accurately identify population trends. In response, monitoring protocols were changed to have a goal of counting lek sites multiple times within a season. We assessed the influence of this change in monitoring protocols on model accuracy and precision applying generalized additive models to describe trends over time. We found that at large spatial scales including >50 leks, the absence of repeated counts within a year did not significantly alter population trend estimates or interpretation. Increasing sample size decreased the model confidence intervals. We developed a population trend model for Wyoming greater sage-grouse from 1965 to 2008, identifying significant changes in the population indices and capturing the cyclic nature of this species. Most sage-grouse declines in Wyoming occurred between 1965 and the 1990s and lek count numbers generally increased from the mid-1990s to 2008. Our results validate the combination of monitoring data collected under different protocols in past and future studies—provided those studies are addressing large-scale questions. We suggest that a larger sample of individual leks is preferable to multiple counts of a smaller sample of leks. © 2011 The Wildlife Society.     

Estimating density of ant nests using distance sampling

The quantification of ant nest densities is a useful but challenging task given the group’s high abundance and diversity of nesting sites. We present a new application of a distance-sampling method which follows standard distance analytical procedures, but introduces a sampling innovation that is particularly useful for ants; instead of having an observer look for ants we let ants find a bait station and measure the distances covered between nest and station. We test this method by estimating the density of epigaeic ant nests in an Amazon tropical forest site near Manaus, Brazil. We distributed 220 baits of canned sardine mixed with cassava flour among 10, 210-m long transects in old-growth upland forest. Forty-five minutes after baiting, we followed the ants’ trails and measured the linear distance between the bait and each nest’s entrance. We then used the freely available program DISTANCE to estimate the number of nests per unit area while accounting for the effect of distance on the probability that a colony will find a bait. There were found 38 species nesting in 287 different colonies, with an estimated 2.66 nests/m². This estimate fell within the 95 % confidence bounds of nest density predicted for a similar number of species based on a literature survey of ant species richness and nest density. Our sampling solution, however, takes less than 30 % of the time used by conventional sampling approaches for a similar area, with the advantage that it produces not only a point estimate but also a quantification of uncertainty about density.     

GCHap: fast MLEs for haplotype frequencies by gene counting

GCHap quickly finds maximum likelihood estimates (MLEs) of frequencies of haplotypes given genotype information on a random sample of individuals. It uses the gene counting method but by excluding haplotypes with zero MLE at an early stage, this implementation uses many orders of magnitude less space and time than naive implementations. A second program, ApproxGCHap, is provided to give alternate estimates for data sets with large numbers of loci or large amounts of missing genotypes. AVAILABILITY: The Java classes and Javadocs pages for GCHap can be obtained from bioinformatics.med.utah.edu/~alun     

Imidacloprid Inhibits Behavioral Defences of the Leaf-Cutting Ant Acromyrmex subterraneus subterraneus (Hymenoptera:Formicidae)

Leaf-cutting ants have evolved a range of defensive strategies which complicate the use of entomopathogens for their control. One of these behavioral strategies is self-grooming, which increases when ants detect the presence of fungal conidia on their integuments. We have previously shown that insecticides, when used at ultra-low concentrations, can be synergists of entomopathogenic fungi. It is possible that certain insecticides could modify ant behavior in a way that increases the chances of a fungal infection taking hold. The current study investigated the effects of the neonicotinoid insecticide Imidacloprid (IMI) and the entomopathogenic fungus Beauveria bassiana on self-grooming and locomotion behavior of Acromrymex subterraneus subterraneus workers when ants were exposed to the agents separately or together. Initially 10, 20 and 40 ng/insect IMI was topically administered to worker ants. Four hours after administration, the ants were placed into an experimental arena and self-grooming and locomotion behaviors were recorded for 10 min. Separate groups of ants were exposed to filter paper discs impregnated with conidial suspensions and 4 h later their behavior was monitored for 10 min. Subsequently, ants were treated with 10 ng/insect IMI, immediately exposed to fungal suspensions and 4 h later their behavior was monitored. The 10 ng/insect IMI treatment resulted in higher locomotor activity and lower self-grooming when compared to vehicle, 20 ng and 40 ng IMI treatments. Exposure to fungal conidia produced low locomotion and high self-grooming behaviors. The 10 ng IMI + fungal conidia treatment resulted in lower self-grooming and higher locomotion behavior. These alterations caused by a low concentration of IMI could increase ant susceptibility to infection by entomopathogenic fungi and are thus of interest for future tests in the development of integrated pest management strategies.     

Is time on our side? Strengthening the link between field efforts and conservation needs

The collection and organization of distributional data is the first crucial stage of any conservation planning action: therefore the decline in field research has implications in both the systematic, floristic and conservation fields. The aim of this paper is to analyze the effects of data updating on conservation planning and priorities. Focusing on the short time frame ranging from 2006 to 2011, we present a case study showing the rate of increase of collected data (taxa and records) and the consequential effects on the definition of areas of priority interest for plant conservation (Important Plant Areas—IPAs). We gathered data on a total of 193 taxa and 849 records with a mean rate of increase of +97 % for taxa and +166 % for records (2006/2011). This increase caused a positive rate of change in high ranking cells (+78 %) defining IPAs, while the number of low ranking cells and no data cells slightly decreased (?12 and ?8 %, respectively). Our results suggest that specific investment to complete the knowledge on the distribution of selected taxa (e.g. 193 taxa represent the 7.5 % of the total vascular flora of Sardinia) would dramatically reduce both the Linnean and Wallacean shortfalls and would allow robust conservation programs to preserve the diversity of the island. Updating the IPAs on a regular basis is a good example of a process that has a low impact as well as a big potential gain especially when field research can only be performed with low intensity and small monetary investments.     

An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [¹H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [¹H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories.     

Testing non-additive effects of nectar-robbing ants and hummingbird pollination on the reproductive success of a parasitic plant

Nectar robbing may have an indirect negative effect on plant reproduction by discouraging legitimate pollinator species from visiting robbed flowers. In this study, we set up a 2 × 2 factorial design with nectar-robbing ants and hummingbird pollination to test for non-additive effects on fruit set, seed mass, and seed germination of the leafless mistletoe Tristerix aphyllus (Loranthaceae). Even though ants caused conspicuous damage at the base of the floral tubes, nectar availability was reduced by only 8 % in the presence of ants. The green-backed firecrown Sephanoides sephaniodes was insensitive to the presence of ants. Rather, the bird responded to flower number and the presence or the absence of damage, but not to the extent of damage within inflorescences. As hummingbirds were largely insensitive to variation in nectar robbing, the interaction ant × hummingbird had no effect on plant-reproductive success. Thus, the factorial experiment did not provide evidence for indirect negative effects of nectar robbing on plant reproduction. These results suggest that indirect effects of nectar robbers on pollinator behaviour may occur under a more restricted set of conditions than those previously considered. We suggest that the low amount of nectar removed by nectar-robbing ants was insufficient for hummingbirds to avoid robbed flowers, which restricted the potential for non-additive effects.     

Estimating density of calling male Eleutherodactylus coqui in Hawaii from audio recordings of the nighttime frog chorus

Analysis of sound pressure data obtained from audio recordings of night-time Eleutherodactylus coqui chorusing was used to estimate population density of calling male coqui frogs in the major Hawaiian island populations. Study plots 400 m² in size (20 by 20 m; n = 32) were established on the islands of Hawaii and Maui to provide venues for field counting calling male frogs to investigate the ground truth relationship with the recorded sound pressure data. Male coqui frogs emit a two-note ‘co-qui’ advertisement call at regular intervals. Exploiting the temperature-related and relatively consistent male coqui call repetition rate and the average emitted ‘co’ call sound pressure level (70.4 dB at 1 m), cue counts obtained from a correlation process available in the program Raven were also used to estimate the density of calling male coqui. The study methodology included comparing linear regression results between the field-counted frogs and the number of frogs obtained from the cue count method. Both methods revealed a linear relationship between calling male population density and associated average sound pressure of the resulting chorus.     

Digital data collection in paleoanthropology

Understanding patterns of human evolution across space and time requires synthesizing data collected by independent research teams, and this effort is part of a larger trend to develop cyber infrastructure and e‐science initiatives. 1 At present, paleoanthropology cannot easily answer basic questions about the total number of fossils and artifacts that have been discovered, or exactly how those items were collected. In this paper, we examine the methodological challenges to data integration, with the hope that mitigating the technical obstacles will further promote data sharing. At a minimum, data integration efforts must document what data exist and how the data were collected (discovery), after which we can begin standardizing data collection practices with the aim of achieving combined analyses (synthesis). This paper outlines a digital data collection system for paleoanthropology. We review the relevant data management principles for a general audience and supplement this with technical details drawn from over 15 years of paleontological and archeological field experience in Africa and Europe. The system outlined here emphasizes free open‐source software (FOSS) solutions that work on multiple computer platforms; it builds on recent advances in open‐source geospatial software and mobile computing.     

Collection and analysis of kinetic data from a stopped-flow spectrophotometer using a microcomputer

A method of interfacing an inexpensive microcomputer to a stopped-flow kinetics spectrophotometer is described. It allows software-selectable sampling frequencies between 0.1 ms and 8 s and large numbers of data points to be collected. Machine language routines to use the interface are described and these allow the sampling frequency to be altered during data collection to ensure adequate numbers of points in critical regions of the kinetic profile. BASIC programs for collection and analysis of multicomponent kinetic data using this system are also described. Due to the large number of data points that can be collected and the ability to selectively sample transmittance values in regions where the signal is rapidly changing with time, relatively unsophisticated methods of data analysis can be used. These methods are suitable for use by microcomputers and mean that data analysis and acquisition can be performed on the same microcomputer in real time. To illustrate this, multicomponent analysis of kinetic transients is performed on simulated data and on the dissociation kinetics of the ethidium-DNA complex.     

Who shall I say is calling? Validation of a caller recognition procedure in Bornean flanged male orangutan (Pongo pygmaeus wurmbii) long calls

Acoustic individual discrimination has been demonstrated for a wide range of animal taxa. However, there has been far less scientific effort to demonstrate the effectiveness of automatic individual identification, which could greatly facilitate research, especially when data are collected via an acoustic localization system (ALS). In this study, we examine the accuracy of acoustic caller recognition in long calls (LCs) emitted by Bornean male orangutans (Pongo pygmaeus wurmbii) derived from two data-sets: the first consists of high-quality recordings taken during individual focal follows (N = 224 LCs by 14 males) and the second consists of LC recordings with variable microphone-caller distances stemming from ALS (N = 123 LCs by 10 males). The LC is a long-distance vocalization. We therefore expect that even the low-quality test-set should yield caller recognition results significantly better than by chance. Automatic individual identification was accomplished using software originally developed for human speaker recognition (i.e. the MSR identity toolbox). We obtained a 93.3% correct identification rate with high-quality recordings, and 72.23% with recordings stemming from the ALS with variable microphone-caller distances (20–420 m). These results show that automatic individual identification is possible even though the accuracy declines compared with the results of high-quality recordings due to severe signal degradations (e.g. sound attenuation, environmental noise contamination, and echo interference) with increasing distance. We therefore suggest that acoustic individual identification with speaker recognition software can be a valuable tool to apply to data obtained through an ALS, thereby facilitating field research on vocal communication.     

Does Colour of the Food Attract Ants?

Milky white, brown, yellow and pink sugar grains along with normal sugar grains in equal number were offered to the ants at different sites, in the foraging ground of a garden locating at Garia, Kolkata, India to note the role of colouration of the food in food selection, if any. It is revealed that the ants Anoplolepis gracilipes procured all the supplied colour sugar grains from the offered sites between 1 and 43 min and the normal sugar grains between 1 and 38 min from the offered sites. Results of statistical analyses clearly indicate that the colour of the food has no role to attract the ants A. gracilipes in respect to procurement times of the sugar grains noted. This suggests that the colour of the food does not act as an attractant for the ants A. gracilipes in food selection.     

Rice diversity panel provides accurate genomic predictions for complex traits in the progenies of biparental crosses involving members of the panel

Key message

Rice breeding programs based on pedigree schemes can use a genomic model trained with data from their working collection to predict performances of progenies produced through rapid generation advancement.

Abstract

So far, most potential applications of genomic prediction in plant improvement have been explored using cross validation approaches. This is the first empirical study to evaluate the accuracy of genomic prediction of the performances of progenies in a typical rice breeding program. Using a cross validation approach, we first analyzed the effects of marker selection and statistical methods on the accuracy of prediction of three different heritability traits in a reference population (RP) of 284 inbred accessions. Next, we investigated the size and the degree of relatedness with the progeny population (PP) of sub-sets of the RP that maximize the accuracy of prediction of phenotype across generations, i.e., for 97 F₅–F₇ lines derived from biparental crosses between 31 accessions of the RP. The extent of linkage disequilibrium was high (r ² = 0.2 at 0.80 Mb in RP and at 1.1 Mb in PP). Consequently, average marker density above one per 22 kb did not improve the accuracy of predictions in the RP. The accuracy of progeny prediction varied greatly depending on the composition of the training set, the trait, LD and minor allele frequency. The highest accuracy achieved for each trait exceeded 0.50 and was only slightly below the accuracy achieved by cross validation in the RP. Our results thus show that relatively high accuracy (0.41–0.54) can be achieved using only a rather small share of the RP, most related to the PP, as the training set. The practical implications of these results for rice breeding programs are discussed.

     

Ant exclusion in citrus over an 8-year period reveals a pervasive yet changing effect of ants on a Mediterranean spider assemblage

Ants and spiders are ubiquitous generalist predators that exert top-down control on herbivore populations. Research shows that intraguild interactions between ants and spiders can negatively affect spider populations, but there is a lack of long-term research documenting the strength of such interactions and the potentially different effects of ants on the diverse array of species in a spider assemblage. Similarly, the suitability of family-level surrogates for finding patterns revealed by species-level data (taxonomic sufficiency) has almost never been tested in spider assemblages. We present a long-term study in which we tested the impact of ants on the spider assemblage of a Mediterranean citrus grove by performing sequential 1-year experimental exclusions on tree canopies for 8 years. We found that ants had a widespread influence on the spider assemblage, although the effect was only evident in the last 5 years of the study. During those years, ants negatively affected many spiders, and effects were especially strong for sedentary spiders. Analyses at the family level also detected assemblage differences between treatments, but they concealed the different responses to ant exclusion shown by some related spider species. Our findings show that the effects of experimental manipulations in ecology can vary greatly over time and highlight the need for long-term studies to document species interactions.     

Relationship between endospore viability and insecticidal potency of Bacillus thuringiensis subsp. aizawai NT0423

Bioassay can be used for analysis of the biological potency of Bacillus thuringiensis (Bt) in fermentation and formulation but requires precise scheduling and several repetitions. Alternatively, this work explored if the endospore counting could be used to predict the potency of Bt technical powder. Analyses of Bt technical powers provided a strong linear relationship (r = 0.971) between the number of viable endospores and the potency of the technical powder against second instar Plutella xylostella (L.) larvae. Next, a Bt wettable powder formulation was stored at 25 and 40 °C for 12 weeks to investigate the influence of storage temperature on the prediction of insecticidal potency based on the counting. At 25 °C storage, the insecticidal potency could be predicted based on the counting, but at 40 °C the predicted insecticidal potency was much lower than the measured potency. These results suggest that the NT0423 endospore viability can be used to predict its potency in production, but the relationship may not be the same following the storage at high temperature.