Automated workflows using Quantitative Colour Pattern Analysis (QCPA): a guide to batch processing and downstream data analysis.

Animal and plant colouration presents a striking dimension of phenotypic variation, the study of which has driven general advances in ecology, evolution, and animal behaviour. Quantitative Colour Pattern Analysis (QCPA) is a dynamic framework for analysing colour patterns through the eyes of non-human observers. However, its extensive array of user-defined image processing and analysis tools means image analysis is often time-consuming. This hinders the full use of analytical power provided by QCPA and its application to large datasets. Here, we offer a robust and comprehensive batch script, allowing users to automate many QCPA workflows. We also provide a complimentary set of useful R scripts for downstream data extraction and analysis. The presented batch processing extension will empower users to further utilise the analytical power of QCPA and facilitate the development of customised semi-automated workflows. Such quantitatively scaled workflows are crucial for exploring colour pattern spaces and developing ever-richer frameworks for analysing organismal colouration accounting for visual perception in animals other than humans. These advances will, in turn, facilitate testing hypotheses on the function and evolution of vision and signals at quantitative and qualitative scales, which are otherwise computationally unfeasible. Supplementary Information: The online version contains supplementary material available at 10.1007/s10682-024-10291-7.

The flashy escape: support for dynamic flash coloration as anti-predator defence.

Dynamic flash coloration is a type of antipredator coloration where intermittently appearing colour patterns in moving animals misdirect predator attacks by obscuring the precise location and trajectory of the moving prey. Birds and butterflies with differing dorsoventral wing coloration or iridescent surface structures may potentially benefit from such effects. However, we lack an understanding of what makes for an effective dynamic flash colour design and how much it benefits the carrier. Here, we test the effect of colour flashing using small passerine birds preying upon colourful, moving, virtual 'prey' stimuli on a touchscreen. We show that at fast speeds, green-to-blue flashing colour patterns can reduce the likelihood of pecks hitting the target, induce greater error in targeting accuracy and increase the number of pecks at a stimulus relative to similarly coloured non-flashing targets. Our results support the idea that dynamic flash coloration can deflect predatory attacks at fast speeds, but the effect may be the opposite when moving slowly.

Deceptive pollinator lures benefit from physical and perceptual proximity to flowers.

Predators often use deception to exploit sensory and cognitive biases in prey. In pollinating insects, these include preferences for conspicuous colours associated with flowers, which predators such as orb-web spiders display as prey lures. Theory predicts that deceptive signal efficacy should covary with both their perceptual similarity and physical proximity to the resources-here, flowers-whose cues they are imitating. Here I used the colour-polymorphic jewelled spider Gasteracantha fornicata to test this prediction. I first examined spiders' capture success in the field, and found their visual resemblance and physical proximity to flowers interacted to mediate capture rates, with colour-similarity becoming increasingly important as the distance between spiders and flowers decreased. I then replicated this interaction experimentally. Spiders adjacent to colour-matched flowers enjoyed heightened capture success relative to those with nearby but colour-mismatched flowers. While spiders with flowers placed at a distance (irrespective of colour) recorded the fewest captures. These results support 'neighbourhood' effects in aggressive deception as receivers' vulnerability to exploitation is mediated by the local signalling community. More generally, they emphasise the importance of the broader information landscape in the ecology of communication, and suggest misinformation is most effective when physically and perceptually proximate to the truth.

Artificial flowers as a tool for investigating multimodal flower choice in wild insects.

Flowers come in a variety of colours, shapes, sizes and odours. Flowers also differ in the quality and quantity of nutritional reward they provide to entice potential pollinators to visit. Given this diversity, generalist flower-visiting insects face the considerable challenge of deciding which flowers to feed on and which to ignore. Working with real flowers poses logistical challenges due to correlations between flower traits, maintenance costs and uncontrolled variables. Here, we overcome this challenge by designing multimodal artificial flowers that varied in visual, olfactory and reward attributes. We used artificial flowers to investigate the impact of seven floral attributes (three visual cues, two olfactory cues and two rewarding attributes) on flower visitation and species richness. We investigated how flower attributes influenced two phases of the decision-making process: the decision to land on a flower, and the decision to feed on a flower. Artificial flowers attracted 890 individual insects representing 15 morphospecies spanning seven arthropod orders. Honeybees were the most common visitors accounting for 46% of visitors. Higher visitation rates were driven by the presence of nectar, the presence of linalool, flower shape and flower colour and was negatively impacted by the presence of citral. Species richness was driven by the presence of nectar, the presence of linalool and flower colour. For hymenopterans, the probability of landing on the artificial flowers was influenced by the presence of nectar or pollen, shape and the presence of citral and/or linalool. The probability of feeding increased when flowers contained nectar. For dipterans, the probability of landing on artificial flowers increased when the flower was yellow and contained linalool. The probability of feeding increased when flowers contained pollen, nectar and linalool. Our results demonstrate the multi-attribute nature of flower preferences and highlight the usefulness of artificial flowers as tools for studying flower visitation in wild insects.

The impact of elevated aestivation temperatures on the behaviour of bogong moths (Agrotis infusa).

Bogong moths are an iconic Australian insect. They migrate annually in spring from low elevation locations in southern Australia to the Australian Alps where they aestivate during summer. As summer ends they make their return journey to the breeding grounds where they mate, lay eggs, and die. Given the moth's extreme behaviour in seeking out cool alpine habitat and with the knowledge that average temperatures at their aestivation sites are rising because of climate change, we first asked whether increased temperatures affect bogong moth activity during aestivation. We found that moth behaviour patterns changed from showing peaks at dawn and dusk with supressed activity during the day at cooler temperatures to near-constant activity at all times of day at 15 °C. Second, we asked whether moth mass changes after aestivating at different temperatures for a week due to dehydration or consumption of body energy reserves. We found that moth wet mass loss increased with increasing temperature, but found no difference in dry mass among temperature treatments. Overall, our results suggest that bogong moth aestivation behaviour changes with temperature and that it may be lost at around 15 °C. The impact of warming on the likelihood of individuals to complete their aestivation in the field should be investigated as a matter of priority to better understand the impact of climate change on the Australian alpine ecosystem.

A synthesis of deimatic behaviour.

Deimatic behaviours, also referred to as startle behaviours, are used against predators and rivals. Although many are spectacular, their proximate and ultimate causes remain unclear. In this review we aim to synthesise what is known about deimatic behaviour and identify knowledge gaps. We propose a working hypothesis for deimatic behaviour, and discuss the available evidence for the evolution, ontogeny, causation, and survival value of deimatic behaviour using Tinbergen's Four Questions as a framework. Our overarching aim is to direct future research by suggesting ways to address the most pressing questions in this field.

Heightened condition dependent expression of structural coloration in the faces, but not wings, of male and female flies.

Structurally colored sexual signals are a conspicuous and widespread class of ornament used in mate choice, though the extent to which they encode information on the quality of their bearers is not fully resolved. Theory predicts that signaling traits under strong sexual selection as honest indicators should evolve to be more developmentally integrated and exaggerated than nonsexual traits, thereby leading to heightened condition dependence. Here, we test this prediction through examination of the sexually dimorphic faces and wings of the cursorial fly Lispe cana. Males and females possess structural UV-white and golden faces, respectively, and males present their faces and wings to females during close-range, ground-based courtship displays, thereby creating the opportunity for mutual inspection. Across a field-collected sample of individuals, we found that the appearance of the faces of both sexes scaled positively with individual condition, though along separate axes. Males in better condition expressed brighter faces as modeled according to conspecific flies, whereas condition scaled with facial saturation in females. We found no such relationships for their wing interference pattern nor abdomens, with the latter included as a nonsexual control. Our results suggest that the structurally colored faces, but not the iridescent wings, of male and female L. cana are reliable guides to individual quality and support the broader potential for structural colors as honest signals. They also highlight the potential for mutual mate choice in this system, while arguing for 1 of several alternate signaling roles for wing interferences patterns among the myriad taxa which bear them.

The exploitation of sexual signals by predators: a meta-analysis.

Sexual signals are often central to reproduction, and their expression is thought to strike a balance between advertising to mates and avoiding detection by predatory eavesdroppers. Tests of the predicted predation costs have produced mixed results, however. Here we synthesized 187 effects from 78 experimental studies in a meta-analytic test of two questions; namely, whether predators, parasites and parasitoids express preferences for the sexual signals of prey, and whether sexual signals increase realized predation risk in the wild. We found that predators and parasitoids express strong and consistent preferences for signals in forced-choice contexts. We found a similarly strong overall increase in predation on sexual signallers in the wild, though here it was modality specific. Olfactory and acoustic signals increased the incidence of eavesdropping relative to visual signals, which experienced no greater risk than controls on average. Variation in outcome measures was universally high, suggesting that contexts in which sexual signalling may incur no cost, or even reduce the incidence of predation, are common. Our results reveal unexpected complexity in a central viability cost to sexual signalling, while also speaking to applied problems in invasion biology and pest management where signal exploitation holds promise for bio-inspired solutions.

Expression of the mono-ADP-ribosyltransferase ART1 by tumor cells mediates immune resistance in non-small cell lung cancer.

Most patients with non-small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)-induced cell death (NICD) of P2X7 receptor (P2X7R)-expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono-adenosine 5'-diphosphate (ADP)-ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R+ CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R+ CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD+-degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell-dependent manner. This was associated with increased infiltration of activated P2X7R+CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.

A TCR mimic monoclonal antibody reactive with the "public" phospho-neoantigen pIRS2/HLA-A*02:01 complex.

Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed "public" cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope's presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01-restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.

Can the Dynamic Colouration and Patterning of Bluelined Goatfish (Mullidae; Upeneichthys lineatus) Be Perceived by Conspecifics?

Bluelined goatfish (Upeneichthys lineatus) exhibit dynamic body colour changes and transform rapidly from a pale, buff/white, horizontally banded pattern to a conspicuous, vertically striped, red pattern when foraging. This red pattern is potentially an important foraging signal for communication with conspecifics, provided that U. lineatus can detect and discriminate the pattern. Using both physiological and behavioural experiments, we first examined whether U. lineatus possess visual pigments with sensitivity to long ("red") wavelengths of light, and whether they can discriminate the colour red. Microspectrophotometric measurements of retinal photoreceptors showed that while U. lineatuslack visual pigments dedicated to the red part of the spectrum, their pigments likely confer some sensitivity in this spectral band. Behavioural colour discrimination experiments suggested that U. lineatuscan distinguish a red reward stimulus from a grey distractor stimulus of variable brightness. Furthermore, when presented with red stimuli of varying brightness they could mostly discriminate the darker and lighter reds from the grey distractor. We also obtained anatomical estimates of visual acuity, which suggest that U. lineatus can resolve the contrasting bands of conspecifics approximately 7 m away in clear waters. Finally, we measured the spectral reflectance of the red and white colouration on the goatfish body. Visual models suggest that U. lineatus can discriminate both chromatic and achromatic differences in body colouration where longer wavelength light is available. This study demonstrates that U. lineatus have the capacity for colour vision and can likely discriminate colours in the long-wavelength region of the spectrum where the red body pattern reflects light strongly. The ability to see red may therefore provide an advantage in recognising visual signals from conspecifics. This research furthers our understanding of how visual signals have co-evolved with visual abilities, and the role of visual communication in the marine environment.

Conspicuous animal signals avoid the cost of predation by being intermittent or novel: confirmation in the wild using hundreds of robotic prey.

Social animals are expected to face a trade-off between producing a signal that is detectible by mates and rivals, but not obvious to predators. This trade-off is fundamental for understanding the design of many animal signals, and is often the lens through which the evolution of alternative communication strategies is viewed. We have a reasonable working knowledge of how conspecifics detect signals under different conditions, but how predators exploit conspicuous communication of prey is complex and hard to predict. We quantified predation on 1566 robotic lizard prey that performed a conspicuous visual display, possessed a conspicuous ornament or remained cryptic. Attacks by free-ranging predators were consistent across two contrasting ecosystems and showed robotic prey that performed a conspicuous display were equally likely to be attacked as those that remained cryptic. Furthermore, predators avoided attacking robotic prey with a fixed, highly visible ornament that was novel at both locations. These data show that it is prey familiarity-not conspicuousness-that determine predation risk. These findings replicated across different predator-prey communities not only reveal how conspicuous signals might evolve in high predation environments, but could help resolve the paradox of aposematism and why some exotic species avoid predation when invading new areas.

Meta-analytic evidence for quantitative honesty in aposematic signals.

The combined use of noxious chemical defences and conspicuous warning colours is a ubiquitous anti-predator strategy. That such signals advertise the presence of defences is inherent to their function, but their predicted potential for quantitative honesty-the positive scaling of signal salience with the strength of protection-is the subject of enduring debate. Here, we systematically synthesized the available evidence to test this prediction using meta-analysis. We found evidence for a positive correlation between warning colour expression and the extent of chemical defences across taxa. Notably, this relationship held at all scales; among individuals, populations and species, though substantial between-study heterogeneity remains unexplained. Consideration of the design of signals revealed that all visual features, from colour to contrast, were equally informative of the extent of prey defence. Our results affirm a central prediction of honesty-based models of signal function and narrow the scope of possible mechanisms shaping the evolution of aposematism. They suggest diverse pathways to the encoding and exchange of information, while highlighting the need for deeper knowledge of the ecology of chemical defences to enrich our understanding of this widespread anti-predator adaptation.

Love at first flight: wing interference patterns are species-specific and sexually dimorphic in blowflies (Diptera: Calliphoridae).

Wing interference patterns (WIPs) are stable structural colours displayed on insect wings which are only visible at specific viewing geometries and against certain backgrounds. These patterns are widespread among flies and wasps, and growing evidence suggests that they may function as species- and sex-specific mating cues in a range of taxa. As such, it is expected that WIPs should differ between species and show clear sexual dimorphisms. However, the true extent to which WIPs vary between species, sexes and individuals is currently unclear, as previous studies have only taken a qualitative approach, without considering how WIPs might be perceived by the insect. Here, we perform the first quantitative analysis of inter- and intra-specific variation in WIPs across seven Australian species of the blowfly genus Chrysomya. Using multispectral digital imaging and a tentative model of blowfly colour vision, we provide quantitative evidence that WIPs are species-specific, highlight that the extent of divergence is greater in males than in females and demonstrate sexual dimorphisms in several species. These data suggest that WIPs have diversified substantially in blowflies as a result of either sexual or ecological selection.

Birds Perceive More Intraspecific Color Variation in Bird-Pollinated Than Bee-Pollinated Flowers.

Pollinator-mediated selection is expected to constrain floral color variation within plant populations. Here, we test for patterns of constraint on floral color variation in 38 bee- and/or hummingbird-pollinated plant species from Colorado, United States. We collected reflectance spectra for at least 15 individuals in each of 1-3 populations of each species (total 78 populations) and modeled perceived color variation in both bee and bird visual spaces. We hypothesized that bees would perceive less intraspecific color variation in bee-pollinated species (vs. bird-pollinated species), and reciprocally, birds would perceive less color variation in bird-pollinated species (vs. bee-pollinated species). In keeping with the higher dimensionality of the bird visual system, birds typically perceived much more color variation than bees, regardless of plant pollination system. Contrary to our hypothesis, bees perceived equal color variation within plant species from the two pollination systems, and birds perceived more color variation in species that they pollinate than in bee-pollinated species. We propose hypotheses to account for the results, including reduced long-wavelength sensitivity in bees (vs. birds), and the ideas that potential categorical color vision in birds and larger cognitive capacities of birds (vs. bees) reduces their potential discrimination against floral color variants in species that they pollinate, resulting in less stabilizing selection on color within bird-pollinated vs. bee-pollinated species.

Macroecological patterns in flower colour are shaped by both biotic and abiotic factors.

There is a wealth of research on the way interactions with pollinators shape flower traits. However, we have much more to learn about influences of the abiotic environment on flower colour. We combine quantitative flower colour data for 339 species from a broad spatial range covering tropical, temperate, arid, montane and coastal environments from 9.25ºS to 43.75ºS with 11 environmental variables to test hypotheses about how macroecological patterns in flower colouration relate to biotic and abiotic conditions. Both biotic community and abiotic conditions are important in explaining variation of flower colour traits on a broad scale. The diversity of pollinating insects and the plant community have the highest predictive power for flower colouration, followed by mean annual precipitation and solar radiation. On average, flower colours are more chromatic where there are fewer pollinators, solar radiation is high, precipitation and net primary production are low, and growing seasons are short, providing support for the hypothesis that higher chromatic contrast of flower colours may be related to stressful conditions. To fully understand the ecology and evolution of flower colour, we should incorporate the broad selective context that plants experience into research, rather than focusing primarily on effects of plant-pollinator interactions.

Photoreflectance/scattering measurements of spider silks informed by standard optics.

The silks of certain orb weaving spiders are emerging as high-quality optical materials. This motivates study of the optical properties of such silk and particularly the comparative optical properties of the silks of different species. Any differences in optical properties may impart biological advantage for a spider species and make the silks interesting for biomimetic prospecting as optical materials. A prior study of the reflectance of spider silks from 18 species reported results for three species of modern orb weaving spiders (Nephila clavipes, Argiope argentata and Micrathena Schreibersi) as having reduced reflectance in the UV range. (Modern in the context used here means more recently derived.) The reduced UV reflectance was interpreted as an adaptive advantage in making the silks less visible to insects. Herein, a standard, experimental technique for measuring the reflectance spectrum of diffuse surfaces, using commercially available equipment, has been applied to samples of the silks of four modern species of orb weaving spiders: Phonognatha graeffei, Eriophora transmarina, Nephila plumipes and Argiope keyserlingi. This is a different technique than used in the previous study. Three of the four silks measured have a reduced signal in the UV. By taking the form of the silks as optical elements into account, it is shown that this is attributable to a combination of wavelength-dependent absorption and scattering by the silks rather than differences in reflectance for the different silks. Phonognatha graeffei dragline silk emerges as a very interesting spider silk with a flat 'reflectance'/scattering spectrum which may indicate it is a low UV absorbing dielectric micro-fibre. Overall the measurement emerges as having the potential to compare the large numbers of silks from different species to prospect for those which have desirable optical properties.

Structural colours reflect individual quality: a meta-analysis.

Colourful ornaments often communicate salient information to mates, and theory predicts covariance between signal expression and individual quality. This has borne out among pigment-based signals, but the potential for 'honesty' in structural coloration is unresolved. Here, I synthesized the available evidence to test this prediction via meta-analysis and found that, overall, the expression of structurally coloured sexual signals is positively associated with individual quality. The effects varied by the measure of quality, however, with body condition and immune function reliably encoded across taxa, but not age nor parasite resistance. The relationship was apparent for both the colour and brightness of signals and was slightly stronger for iridescent ornaments. These results suggest diverse pathways to the encoding and exchange of information among structural colours while highlighting outstanding questions as to the development, visual ecology and evolution of this striking adornment.

Flies Exploit Predictable Perspectives and Backgrounds to Enhance Iridescent Signal Salience and Mating Success.

Communication requires both the encoding of information and its effective transmission, but little is known about display traits that primarily serve to enhance efficacy. Here we examined the visual courtships of Lispe cana, a cursorial fly that lives and mates in heterogeneous foreshores, and tested the prediction that males should seek to enhance signal salience and consequent fitness through the flexible choice of display locations. We show that courting males access the field of view of females by straddling them and holding their wings closed before moving ahead to present their structurally colored faces in ritualized dances. Males preferentially present these UV-white signals against darker backgrounds and the magnitude of contrast predicts female attention, which in turn predicts mating success. Our results demonstrate a striking interplay between the physical and attentional manipulation of receivers and reveal novel routes to the enhancement of signal efficacy in noisy environments.