Innes C. Cuthill

Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research

animals used (i.e., species/strain, sex, and age/weight). Most of the papers surveyed did not report using randomisation (87%) or blinding (86%) to reduce bias in animal selection and outcome assessment. Only 70% of the publications that used statistical methods fully described them and presented the results with a measure of precision or variability [5]. These findings are a cause for concern and are consistent with reviews of many research areas, including clinical studies, published in recent years [2–22].

Animal research: Reporting in vivo experiments: The ARRIVE guidelines

2 Provide an accurate summary of the background, research objectives, including details of the species or strain of animal used, key methods, principal findings and conclusions of the study. INTRODUCTION Background 3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale. b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study’s relevance to human biology. Objectives 4 Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested. METHODS Ethical statement 5 Indicate the nature of the ethical review permissions, relevant licences (e.g. Animal [Scientific Procedures] Act 1986), and national or institutional guidelines for the care and use of animals, that cover the research. Study design 6 For each experiment, give brief details of the study design including: a. The number of experimental and control groups. b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when). c. The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out. Experimental procedures 7 For each experiment and each experimental group, including controls, provide precise details of all procedures carried out. For example: a. How (e.g. drug formulation and dose, site and route of administration, anaesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s). b. When (e.g. time of day). c. Where (e.g. home cage, laboratory, water maze). d. Why (e.g. rationale for choice of specific anaesthetic, route of administration, drug dose used). BJP British Journal of Pharmacology DOI:10.1111/j.1476-5381.2010.00872.x www.brjpharmacol.org British Journal of Pharmacology (2010) 16

Effect size, confidence interval and statistical significance: a practical guide for biologists

Null hypothesis significance testing (NHST) is the dominant statistical approach in biology, although it has many, frequently unappreciated, problems. Most importantly, NHST does not provide us with two crucial pieces of information: (1) the magnitude of an effect of interest, and (2) the precision of the estimate of the magnitude of that effect. All biologists should be ultimately interested in biological importance, which may be assessed using the magnitude of an effect, but not its statistical significance. Therefore, we advocate presentation of measures of the magnitude of effects (i.e. effect size statistics) and their confidence intervals (CIs) in all biological journals. Combined use of an effect size and its CIs enables one to assess the relationships within data more effectively than the use of p values, regardless of statistical significance. In addition, routine presentation of effect sizes will encourage researchers to view their results in the context of previous research and facilitate the incorporation of results into future meta‐analysis, which has been increasingly used as the standard method of quantitative review in biology. In this article, we extensively discuss two dimensionless (and thus standardised) classes of effect size statistics: d statistics (standardised mean difference) and r statistics (correlation coefficient), because these can be calculated from almost all study designs and also because their calculations are essential for meta‐analysis. However, our focus on these standardised effect size statistics does not mean unstandardised effect size statistics (e.g. mean difference and regression coefficient) are less important. We provide potential solutions for four main technical problems researchers may encounter when calculating effect size and CIs: (1) when covariates exist, (2) when bias in estimating effect size is possible, (3) when data have non‐normal error structure and/or variances, and (4) when data are non‐independent. Although interpretations of effect sizes are often difficult, we provide some pointers to help researchers. This paper serves both as a beginner’s instruction manual and a stimulus for changing statistical practice for the better in the biological sciences.

Tetrachromacy, oil droplets and bird plumage colours

Abstract There is a growing body of data on avian eyes, including measurements of visual pigment and oil droplet spectral absorption, and of receptor densities and their distributions across the retina. These data are sufficient to predict psychophysical colour discrimination thresholds for light-adapted eyes, and hence provide a basis for relating eye design to visual needs. We examine the advantages of coloured oil droplets, UV vision and tetrachromacy for discriminating a diverse set of avian plumage spectra under natural illumination. Discriminability is enhanced both by tetrachromacy and coloured oil droplets. Oil droplets may also improve colour constancy. Comparison of the performance of a pigeons eye, where the shortest wavelength receptor peak is at 410 nm, with that of the passerine Leiothrix, where the ultraviolet-sensitive peak is at 365 nm, generally shows a small advantage to the latter, but this advantage depends critically on the noise level in the sensitivity mechanism and on the set of spectra being viewed.

Survey of the quality of experimental design, statistical analysis and reporting of research using animals.

For scientific, ethical and economic reasons, experiments involving animals should be appropriately designed, correctly analysed and transparently reported. This increases the scientific validity of the results, and maximises the knowledge gained from each experiment. A minimum amount of relevant information must be included in scientific publications to ensure that the methods and results of a study can be reviewed, analysed and repeated. Omitting essential information can raise scientific and ethical concerns. We report the findings of a systematic survey of reporting, experimental design and statistical analysis in published biomedical research using laboratory animals. Medline and EMBASE were searched for studies reporting research on live rats, mice and non-human primates carried out in UK and US publicly funded research establishments. Detailed information was collected from 271 publications, about the objective or hypothesis of the study, the number, sex, age and/or weight of animals used, and experimental and statistical methods. Only 59% of the studies stated the hypothesis or objective of the study and the number and characteristics of the animals used. Appropriate and efficient experimental design is a critical component of high-quality science. Most of the papers surveyed did not use randomisation (87%) or blinding (86%), to reduce bias in animal selection and outcome assessment. Only 70% of the publications that used statistical methods described their methods and presented the results with a measure of error or variability. This survey has identified a number of issues that need to be addressed in order to improve experimental design and reporting in publications describing research using animals. Scientific publication is a powerful and important source of information; the authors of scientific publications therefore have a responsibility to describe their methods and results comprehensively, accurately and transparently, and peer reviewers and journal editors share the responsibility to ensure that published studies fulfil these criteria.

Plumage Reflectance and the Objective Assessment of Avian Sexual Dichromatism

Assessment of color using human vision (or standards based thereon) is central to tests of many evolutionary hypotheses. Yet fundamental differences in color vision between humans and other animals call this approach into question. Here we use techniques for objectively assessing color patterns that avoid reliance on species‐specific (e.g., human) perception. Reflectance spectra are the invariant features that we expect the animals color cognition to have evolved to extract. We performed multivariate analyses on principal components derived from >2,600 reflectance spectra (300–720 nm) sampled in a stratified random design from different body regions of male and female starlings in breeding plumage. Starlings possess spatially complex plumage patterns and extensive areas of iridescence. Our study revealed previously unnoticed sex differences in plumage coloration and the nature of iridescent and noniridescent sex differences. Sex differences occurred in some body regions but not others, were more pronounced at some wavelengths (both ultraviolet and human visible), and involved differences in mean reflectance and spectral shape. Discriminant analysis based on principal components were sufficient to sex correctly 100% of our sample. If hidden sexual dichromatism is widespread, then it has important implications for classifications of animals as mono‐ or dimorphic and for taxonomic and conservation purposes.

Ultraviolet Vision in Birds

Publisher Summary Birds can see ultraviolet (UV) light because, unlike humans, their lenses and other ocular media transmit UV, and they possess a class of photoreceptor, which is maximally sensitive to violet or UV light, depending on the species. Birds have a tetrachromatic color space, as compared to the trichromacy of humans. Birds, along with some reptiles and fish, also possess double cones in large numbers and a cone class. This chapter discusses a range of behavioral experiments, from several species, which show that UV information is utilized in behavioral decisions, notably in foraging and signaling. Removal of UV wavelengths affects mate choice even in species that are colorful to humans. These studies emphasize that avian and human color perceptions are different and that the use of human color standards, and even artificial lighting, may produce misleading results. However, genuinely objective measures of color are available, as are, importantly, models for mapping the measured spectra into an avian color space.

Visual pigments, oil droplets, ocular media and cone photoreceptor distribution in two species of passerine bird: the blue tit (Parus caeruleus L.) and the blackbird (Turdus merula L.)

Abstract The spectral absorption characteristics of the retinal photoreceptors of the blue tit (Parus caeruleus) and blackbird (Turdus merula) were investigated using microspectrophotometry. The retinae of both species contained rods, double cones and four spectrally distinct types of single cone. Whilst the visual pigments and cone oil droplets in the other receptor types are very similar in both species, the wavelength of maximum sensitivity (λmax) of long-wavelength-sensitive single and double cone visual pigment occurs at a shorter wavelength (557 nm) in the blackbird than in the blue tit (563 nm). Oil droplets located in the long-wavelength-sensitivesingle cones of both species cut off wavelengths below 570–573 nm, theoretically shifting cone peak spectral sensitivity some 40 nm towards the long-wavelength end of the spectrum. This raises the possibility that the precise λmax of the long-wavelength-sensitive visual pigment is optimised for the visual function of the double cones. The distribution of cone photoreceptors across the retina, determined using conventional light and fluorescence microscopy, also varies between the two species and may reflect differences in their visual ecology.

Disruptive coloration and background pattern matching

Effective camouflage renders a target indistinguishable from irrelevant background objects. Two interrelated but logically distinct mechanisms for this are background pattern matching (crypsis) and disruptive coloration: in the former, the animals colours are a random sample of the background; in the latter, bold contrasting colours on the animals periphery break up its outline. The latter has long been proposed as an explanation for some apparently conspicuous coloration in animals, and is standard textbook material. Surprisingly, only one quantitative test of the theory exists, and one experimental test of its effectiveness against non-human predators. Here we test two key predictions: that patterns on the bodys outline should be particularly effective in promoting concealment and that highly contrasting colours should enhance this disruptive effect. Artificial moth-like targets were exposed to bird predation in the field, with the experimental colour patterns on the ‘wings’ and a dead mealworm as the edible ‘body’. Survival analysis supported the predictions, indicating that disruptive coloration is an effective means of camouflage, above and beyond background pattern matching.

Sexual Selection and the Mismeasure of Color

Many long-standing evolutionary hypotheses make predictions about trends in color patterns. Examples of these include crypsis, mimicry and warning coloration, fruit coloration, flower coloration, the handicap principle of honest advertisement, Fishers runaway process, the parasite theory of sexual selection, and sensory drive theories of signaling. The majority of tests of these hypotheses, particularly with regard to sexual selection, have been conducted on objects that birds perceive visually, with human vision used to assess color. This assumes that birds see color patterns as humans do, an assumption that is seriously flawed. First, birds see very well parts of the spectrum that humans cannot. Second, birds have at least four dimensions to their color vision, compared to only three in humans. Third, birds have a complex system of oil droplets in their retinas, which may alter the number of hues they perceive. Thus, an object will not appear to have the same hues for a human and a bird, and maybe not even the same relative brightness or saturation. Despite this, human vision is routinely, and almost without exception, the method used for assessment of color patterns seen by birds. We argue that the error in this assumption may well be a major reason that support for various evolutionary hypotheses involving color is an area of controversy. We also suggest methods for overcoming the shortcomings of existing studies and suggest which previous approaches are likely to have been most erroneous. As part of this, it is apparent that a research program in color cognition is necessary, for if we wish to understand evolutionary hypotheses involving color we need to understand how animals perceive color. Color is not an inherent property of the object; it is a product of the brain of the animal perceiving the object. Humans can see hues seen by birds as much as knowledge of x and y coordinates can predict the position of a point in three-dimensional space.