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.

Chemical defences indicate bold colour patterns with reduced variability in aposematic nudibranchs.

The selective factors that shape phenotypic diversity in prey communities with aposematic animals are diverse and coincide with similar diversity in the strength of underlying secondary defences. However, quantitative assessments of colour pattern variation and the strength of chemical defences in assemblages of aposematic species are lacking. We quantified colour pattern diversity using quantitative colour pattern analysis (QCPA) in 13 dorid nudibranch species (Infraorder: Doridoidei) that varied in the strength of their chemical defences. We accounted for the physiological properties of a potential predator's visual system (a triggerfish, Rhinecanthus aculeatus) and modelled the appearance of nudibranchs from multiple viewing distances (2 and 10 cm). We identified distinct colour pattern properties associated with the presence and strength of chemical defences. Specifically, increases in chemical defences indicated increases in colour pattern boldness (i.e. visual contrast elicited via either or potentially coinciding chromatic, achromatic and/or spatial contrast). Colour patterns were also less variable among species with chemical defences when compared to undefended species. Our results indicate correlations between secondary defences and diverse, bold colouration while showing that chemical defences coincide with decreased colour pattern variability among species. Our study suggests that complex spatiochromatic properties of colour patterns perceived by potential predators can be used to make inferences on the presence and strength of chemical defences.

Signal detectability and boldness are not the same: the function of defensive coloration in nudibranchs is distance-dependent.

Aposematic signals visually advertise underlying anti-predatory defences in many species. They should be detectable (e.g. contrasting against the background) and bold (e.g. using internal pattern contrast) to enhance predator recognition, learning and memorization. However, the signalling function of aposematic colour patterns may be distance-dependent: signals may be undetectable from a distance to reduce increased attacks from naïve predators but bold when viewed up close. Using quantitative colour pattern analysis, we quantified the chromatic and achromatic detectability and boldness of colour patterns in 13 nudibranch species with variable strength of chemical defences in terms of unpalatability and toxicity, approximating the visual perception of a triggerfish (Rhinecanthus aculeatus) across a predation sequence (detection to subjugation). When viewed from an ecologically relevant distance of 30 cm, there were no differences in detectability and boldness between well-defended and undefended species. However, when viewed at closer distances (less than 30 cm), well-defended species were more detectable and bolder than undefended species. As distance increased, detectability decreased more significantly than boldness for defended species. For undefended species, boldness and detectability remained comparatively consistent, regardless of viewing distance. We provide evidence for distance-dependent signalling in aposematic nudibranchs and highlight the importance of distinguishing signal detectability from boldness in studies of aposematism.

Using colour pattern edge contrast statistics to predict detection speed and success in triggerfish (Rhinecanthus aculeatus).

Edge detection is important for object detection and recognition. However, we do not know whether edge statistics accurately predict the detection of prey by potential predators. This is crucial given the growing availability of image analysis software and their application across non-human visual systems. Here, we investigated whether Boundary Strength Analysis (BSA), Local Edge Intensity Analysis (LEIA) and the Gabor edge disruption ratio (GabRat) could predict the speed and success with which triggerfish (Rhinecanthus aculeatus) detected patterned circular stimuli against a noisy visual background, in both chromatic and achromatic presentations. We found various statistically significant correlations between edge statistics and detection speed depending on treatment and viewing distance; however, individual pattern statistics only explained up to 2% of the variation in detection time, and up to 6% when considering edge statistics simultaneously. We also found changes in fish response over time. While highlighting the importance of spatial acuity and relevant viewing distances in the study of visual signals, our results demonstrate the importance of considering explained variation when interpreting colour pattern statistics in behavioural experiments. We emphasize the need for statistical approaches suitable for investigating task-specific predictive relationships and ecological effects when considering animal behaviour. This is particularly important given the ever-increasing dimensionality and size of datasets in the field of visual ecology.

Weapons or deterrents? Nudibranch molluscs use distinct ecological modes of chemical defence against predators.

Defensive chemicals are used by plants and animals to reduce the risk of predation through different mechanisms, including toxins that cause injury and harm (weapons) and unpalatable or odiferous compounds that prevent attacks (deterrents). However, whether effective defences are both toxins and deterrents, or work in just one modality is often unclear. In this study, our primary aim was to determine whether defensive compounds stored by nudibranch molluscs acted as weapons (in terms of being toxic), deterrents (in terms of being distasteful) or both. Our secondary aim was to investigate the response of different taxa to these defensive compounds. To do this, we identified secondary metabolites in 30 species of nudibranch molluscs and investigated their deterrent properties using antifeedant assays with three taxa: rock pool shrimp, Palaemon serenus, and two fish species: triggerfish Rhinecanthus aculeatus and toadfish Tetractenos hamiltoni. We compared these results to toxicity assays using brine shrimp Artemia sp. and previously published toxicity data with a damselfish Chromis viridis. Overall, we found no clear relationship between palatability and toxicity, but instead classified defensive compounds into the following categories: Class I & II-highly unpalatable and highly toxic; Class I-weakly unpalatable and highly toxic; Class II-highly unpalatable but weakly toxic; WR (weak response)-weakly unpalatable and weakly toxic. We also found eight extracts from six species that did not display activity in any assays indicating they may have very limited chemical defensive mechanisms (NR, no response). We found that the different classes of secondary metabolites were similarly unpalatable to fish and shrimp, except extracts from Phyllidiidae nudibranchs (isonitriles) that were highly unpalatable to shrimp but weakly unpalatable to fish. Our results pave the way towards better understanding how animal chemical defences work against a variety of predators. We highlight the need to disentangle weapons and deterrents in future work on anti-predator defences to better understand the foraging decisions faced by predators, the resultant selection pressures imposed on prey and the evolution of different anti-predator strategies.

The Validity of Brine Shrimp (Artemia Sp.) Toxicity Assays to Assess the Ecological Function of Marine Natural Products.

Many organisms employ toxic compounds for protection against predators. To understand the effectiveness of such compounds, chemoecological studies often use brine shrimp (Artemia spp.) as a model organism instead of more ecologically relevant species. This is mostly because brine shrimp assays are simple and quick, but also due to the ethical implications associated with inducing harm to vertebrate predators in toxicity assays. In this study, we examined whether brine shrimp assays produce similar results to ichthyological toxicity assays with the aim of validating the use of brine shrimp as a preliminary screening tool. We extracted compounds from eight nudibranch molluscs including six species that we consider to signal their chemical defenses via warning coloration to visually hunting vertebrate predators. We tested the relative toxicity of these compounds against brine shrimp and a vertebrate potential predator, the blue-green damselfish (Chromis viridis). We found that extracts toxic to brine shrimp were also toxic to damselfish; however, extracts non-toxic to brine shrimp may still be toxic to damselfish. We also produced and tested mantle vs whole-body extracts for some nudibranch species, which exhibited similar toxicities in both assays except for the whole-body extract of Goniobranchus splendidus which was harmless to shrimp but toxic to fish, while the mantle extract was toxic to both. Overall, we argue that the brine shrimp assay can reasonably indicate the potential toxicity of a compound to fish, but additional experiments with more ecologically relevant predators are required if a no dose-response is observed against brine shrimp.

More than noise: context-dependent luminance contrast discrimination in a coral reef fish (Rhinecanthus aculeatus).

Achromatic (luminance) vision is used by animals to perceive motion, pattern, space and texture. Luminance contrast sensitivity thresholds are often poorly characterised for individual species and are applied across a diverse range of perceptual contexts using over-simplified assumptions of an animal's visual system. Such thresholds are often estimated using the receptor noise limited model (RNL). However, the suitability of the RNL model to describe luminance contrast perception remains poorly tested. Here, we investigated context-dependent luminance discrimination using triggerfish (Rhinecanthus aculeatus) presented with large achromatic stimuli (spots) against uniform achromatic backgrounds of varying absolute and relative contrasts. 'Dark' and 'bright' spots were presented against relatively dark and bright backgrounds. We found significant differences in luminance discrimination thresholds across treatments. When measured using Michelson contrast, thresholds for bright spots on a bright background were significantly higher than for other scenarios, and the lowest threshold was found when dark spots were presented on dark backgrounds. Thresholds expressed in Weber contrast revealed lower thresholds for spots darker than their backgrounds, which is consistent with the literature. The RNL model was unable to estimate threshold scaling across scenarios as predicted by the Weber-Fechner law, highlighting limitations in the current use of the RNL model to quantify luminance contrast perception. Our study confirms that luminance contrast discrimination thresholds are context dependent and should therefore be interpreted with caution.

Intertidal gobies acclimate rate of luminance change for background matching with shifts in seasonal temperature.

Rate of colour change and background matching capacity are important functional traits for avoiding predation and hiding from prey. Acute changes in environmental temperature are known to impact the rate at which animals change colour, and therefore may affect their survival. Many ectotherms have the ability to acclimate performance traits such as locomotion, metabolic rate and growth rate with changes in seasonal temperature. However, it remains unclear how other functional traits that are directly linked to behaviour and survival respond to long-term changes in temperature (within an individual's lifetime). We assessed whether the rate of colour change is altered by long-term changes in temperature (seasonal variation) and if rate of colour change can acclimate to seasonal thermal conditions. We used an intertidal rock-pool goby Bathygobius cocosensis, to test this and exposed individuals to representative seasonal mean temperatures (16 or 31°C, herein referred to cold- and warm-exposed fish respectively) for 9 weeks and then tested their rate of luminance change when placed on white and black backgrounds at acute test temperatures 16 and 31°C. We modelled rate of luminance change using the visual sensitives of a coral trout Plectropmus leopardus to determine how well gobies matched their backgrounds in terms of luminance contrast to a potential predator. After exposure to long-term seasonal conditions, the warm-exposed fish had faster rates of luminance change and matched their background more closely when tested at 31 than at 16°C. Similarly, the cold-exposed fish had faster rates of luminance change and matched their backgrounds more closely at 16°C than at 31°C. This demonstrates that rate of luminance change can be adjusted to compensate for long-term changes in seasonal temperature. This is the first study to show that animals can acclimate rate of colour change for background matching to seasonal thermal conditions. We also show that rapid changes in acute temperature reduce background matching capabilities. Stochastic changes in climate are likely to affect the frequency of predator-prey interactions which may have substantial knock-on effects throughout ecosystems.

Toxicity and taste: unequal chemical defences in a mimicry ring.

Mimicry of warning signals is common, and can be mutualistic when mimetic species harbour equal levels of defence (Müllerian), or parasitic when mimics are undefended but still gain protection from their resemblance to the model (Batesian). However, whether chemically defended mimics should be similar in terms of toxicity (i.e. causing damage to the consumer) and/or unpalatability (i.e. distasteful to consumer) is unclear and in many studies remains undifferentiated. In this study, we investigated the evolution of visual signals and chemical defences in a putative mimicry ring of nudibranch molluscs. First, we demonstrated that the appearance of a group of red spotted nudibranchs molluscs was similar from the perspective of potential fish predators using visual modelling and pattern analysis. Second, using phylogenetic reconstruction, we demonstrated that this colour pattern has evolved multiple times in distantly related individuals. Third, we showed that these nudibranchs contained different chemical profiles used for defensive purposes. Finally, we demonstrated that although levels of distastefulness towards Palaemon shrimp remained relatively constant between species, toxicity levels towards brine shrimp varied significantly. We highlight the need to disentangle toxicity and taste when considering chemical defences in aposematic and mimetic species, and discuss the implications for aposematic and mimicry signal evolution.