1 Introduction

Predation is a major threat for flying insects, especially in the tropics where insectivorous birds are diverse and abundant (Haffer 1985; Stotz et al. 1996). While pursuing a given flying prey, both coloration and flight patterns are important factors defining whether the bird will attack (Cott 1940; Kassarov 2003). Therefore, defense mechanisms emerged in several groups where the prey's behavior and coloration operate together to avoid predation (Ruxton, Sherratt, and Speed 2004; Stevens and Ruxton 2019). This is the case of the defense mechanism known as flash coloration.

Flash coloration was described in detail by Cott (1940), who pointed out that some prey animals exhibit a predominantly cryptic pattern when motionless, suddenly revealing a body part of conspicuous color as they move. In essence, crypsis and conspicuousness occur sequentially as the animal alternates between motionless and movement, creating a succession of flashes of conspicuous coloration that may confuse or disorient predators while chasing the prey. According to Edmunds (1974), the appearance of a conspicuous color could make the predator seek the color itself during pursuit, and with the sudden disappearance of the color as the prey becomes motionless, the predator would assume that the prey itself had also disappeared.

Both vertebrates and invertebrates may exhibit contrasting patterns that alternate between cryptic and conspicuous colors during movement (Edmunds 1974). Among insects, numerous examples are found in Lepidoptera, Orthoptera and Phasmatodea, which display conspicuously colored hindwings during movement (Cott 1940; Edmunds 1974).

The defensive value of flash coloration against predation, however, is yet to be demonstrated in nature (Edmunds 2008). Recent experiments using computer simulations and humans as predators have provided important data and insights on the flash coloration hypothesis (Loeffler-Henry et al. 2018; Murali 2018; Bae et al. 2019; Murali and Kodandaramaiah 2020; Loeffler-Henry, Kang, and Sherratt 2021; Sherratt and Loeffler-Henry 2022; Silvasti et al. 2024). However, demonstration of the selective value of flash coloration in nature awaits further experimental manipulation using real predators and real prey.

Due to the great diversity of color patterns in their wings and the ease with which they may be observed and manipulated; butterflies have been extensively used as model organisms in studies of defensive coloration (Edmunds 1974; Brakefield and French 1993). Indeed, part of the huge variation in butterfly color patterns is thought to have evolved as a form of defense against predators, mostly birds (Carpenter 1939; Chai 1986; Pinheiro and Cintra 2017; Willmott et al. 2017).

Tropical Morpho butterflies are well-known for their iridescent structural blue color, with most species presenting contrasting color patterns during flight (Debat et al. 2018). This iridescent color produces very bright, dynamic, directionally reflected signals that change appearance with lighting and viewing angle (Pegram, Han, and Rutowski 2015), which allows these structural colors to carry additional signals than colors based on pigments (Kemp and Rutowski 2007). Widely distributed in Neotropical forests, Morpho helenor (Cramer) alternates dramatically the brightness and spectral properties of their radiance during flight: the butterflies show an iridescent blue band on the dorsal wing surface and a brown pattern with marginal eyespots on the ventral surface, that makes the butterfly cryptic during flight in the dark forest understory (Figure 1). Combined with its erratic flight pattern (Debat et al. 2018), the flashing blue on the dorsal wing surface of M. helenor would make it difficult for a predator to know where the butterfly will move next, as predicted by the flash coloration hypothesis.

Here, we test the flash coloration hypothesis in M. helenor through field experiments in the Brazilian Atlantic rainforest. During its irregular flight in the dark forest understory, we hypothesize that the blue flashes from M. helenor's dorsal wing surface would make it harder for avian predators to predict the insect's flightpath, and thus to successfully capture the butterfly. Specifically, we evaluated butterfly survivorship through time on treated (altered color pattern) versus control (unaltered color pattern) individuals via mark-recapture censuses. Additionally, we manipulated the color of the wing ventral surface to evaluate predation by birds on motionless butterflies pinned on the leaflitter (altered vs. unaltered color pattern), simulating their resting posture (wings closed) on the forest floor.

By experimentally change the dynamic flashing pattern during flight, we were able to demonstrate decreased survival in altered compared to unaltered butterflies. Moreover, butterfly predation on the forest floor proved scant and did not differ between experimental butterfly groups. Our field study with M. helenor provides the first, to our knowledge, experimental evidence in nature supporting flash coloration during flight as a defense mechanism against avian predation in tropical butterflies.

2 Material and Methods

2.3 Experimental Procedures

Color pattern manipulation was performed with non-toxic enamel paint (Testors Co., Rockford, Illinois), which has proven an effective marking method. The paint is waterproof, dries quickly, and has no apparent effect on flight behavior (Simon and Bissinger 1983; Cakmak 2009; Young 2017). To test whether the color of the paints used in the experiments resembled the original colors of the butterfly wing, we measured the reflectance spectrum of individuals with painted wings (10 individuals, 5 for each paint color) and of unpainted individuals (5 individuals to test the blue color and 5 to test the brown color; Figure S3). The reflectance spectrum was measured between 300 and 700 mm (including ultraviolet light [UV]), using a Jaz-S spectrometer (Ocean Optics, Jaz Modular Optical Sensing Suite, Dunedin, FL, USA). A pulsed xenon light source was used with a spectral range between 190 and 1100 nM. A black fiber-optic holder was used to block any external light. Reflectance was evaluated using a PTFE disc (WS-1 Diffuse Reflectance Standard, PTFE; Ocean Optics) as the white standard and a black suede paper as the black standard. The measures were obtain using a standardized region of each individual forewing, which were positioned in a black suede paper horizontally. The spectral analyses were performed using the Pavo package (Maia et al. 2019) in R software (R Core Team 2020).

Because the blue coloration on the dorsal surface is structural and iridescent, the predator's perception of the color changes depending on the butterfly's position and the light conditions. It would be impossible to replicate this pattern on the ventral surface of the butterflies using only paint, as the physical properties are different. However, by adding this metallic blue paint, we guarantee that the butterfly will not display the low reflectance typical of brown coloration during flight (Figure S3). By increasing the reflectance on the ventral surface with blue paint, the experiment reduces the contrast between the ventral and dorsal face of the butterfly, thereby diminishing the confusion effect caused by dynamic coloration. In all experiments, butterflies were assigned to treatment or control group through the flip of a coin (see below). The experiments were carried out during the population peaks of M. helenor at the study site. Experiment 1 lasted 30 days between September and October 2021 and Experiment 2 lasted 30 days between February and March 2021. Experiment 3 was carried out in February/March 2022 and September/October 2023; butterflies were placed on the ground in successive days in each period, totaling 24 days of exposure to predation.

2.3.1 Experiment 1

The manipulation consisted of adding a blue band (color code 1539TT) to the ventral wing surface of each wing, such that flying butterflies in the treatment group would exhibit a bright blue color on both sides of their wings (Figure 2a), thus decreasing the amplitude of the spectral and brightness changes in the butterfly's appearance that occur in flight. Butterflies in the control group were painted with a dark brown band (color code 1121TT) on the same region of each ventral wing, as done in treated individuals (Figure 2b). The eyespot region present on the ventral surface of the wings was kept unpainted in both groups. Because eyespots on the wing can act as a visual signal (Stevens 2005), their removal could affect the experiment. Painted butterflies were kept with their wings open for at least 2 min to ensure the paint dried, and to prevent smudging and sticking.

2.3.2 Experiment 2

Removal of the bright blue band on the dorsal wing surface such that flying butterflies would always be cryptic for a chasing bird (Figure 2c) and, therefore, less vulnerable to predation in the shaded forest understory. In the treatment group, the bright blue band of the dorsal wing surface was covered by a dark brown band (color “Rubber”, code 1183TT, Testors Co., Rockford, Illinois) (Figure 2c). In the control group, a similar dark brown band was painted on the dorsal wing surface of the butterflies next to the bright blue band, thus keeping the dorsal flashing blue during flight (Figure 2d). As in Experiment 1, painted butterflies were kept with their wings open for at least 2 min before release.

2.3.3 Experiment 3

The brown coloration of the ventral wing surface makes M. helenor cryptic when resting on the leaflitter with the wings closed (Figure S2). Therefore, by changing the color pattern of the ventral wing surface by adding a blue band (as in Experiment 1) resting butterflies would become more conspicuous (Figure 3). Experiment 3 was designed to evaluate how the ventral blue bands of blue paint affected the predatory attacks on landed butterflies. Butterflies were manipulated in the same way as in Experiment 1, that is, treatment individuals were painted a blue band on the ventral wing surface whereas control individuals were painted a brown band on the same region of the ventral wing (Figure 2). Butterflies were killed by pinching their thorax using the thumb and forefinger. Individuals were then painted, attached to a wooden stick, and positioned close to the ground at the edge of a forest trail (Figure 3; method follows Willmott et al. 2017, to test predation by birds). We used instant glue (Tekbond) to keep the butterfly wings closed, simulating a resting posture (Figure S2). We applied Tanglefoot resin (Grand Rapids, MI, USA) in the base of the sticks to prevent ground foraging ants from accessing the butterflies. Experimental individuals were positioned at least 10 m from one another, along a 4 km trail in the forest. The direction in which each individual was positioned (North, South, East, or West) was randomly selected. Butterflies in the treatment and control groups were checked for beak marks on their wings every 24 h (around 16:00 h). After checking, experimental butterflies were repositioned along the trail. Beak marks on butterfly wings have a typical triangular shape (bird beak) and have previously been used extensively as indicators of predation attempts by birds (Figure S1; Benson 1972; Shapiro 1974; Bowers and Wiernasz 1979; Ohsaki 1995; Ide 2005).

3 Results

3.1 Experiment 1

The number of recaptured individuals differed significantly between experimental groups of Morpho helenor (Figure 4a). Out of 135 individuals marked in the treatment group (color pattern altered), 20.7% were recaptured. In the control group (color pattern unaltered), out of 134 marked individuals, 37.3% were recaptured. The sex ratio of marked individuals did not differ between experimental groups (χ² = 0.141; df = 1; p = 0.7078).

3.3 Experiment 3

Experimental manipulation used dead butterflies pinned on the leaflitter. We evaluate if treatment butterflies painted with a bright blue band on the ventral wing surface would be more attacked by birds, when landed on the forest floor (wings closed), than control butterflies with unaltered color pattern (brown band added to the ventral wing surface). Avian predation on Morpho helenor butterflies pinned on the forest floor, as expressed by beak marks on their wings, was scant and did not differ between treatment (13.5%, altered individuals) and control (16.5%, unaltered individuals) groups (Figure 5; χ² = 0.47; df = 1; p = 0.49).

4 Discussion

Our study shows that individuals painted with a blue band on the ventral wing surface do not suffer increased predation when landed on the forest floor, which is an indication that this extra blue band does not make the landed butterflies more attractive to predators. Therefore, the decreased survival of treatment compared to control butterflies in Experiment 1 is probably due to predation during flight. Birds are among the main visually oriented predators of butterflies (Théry and Gomez 2010; Carpenter 1939; Chai 1986; Curio 2012), and most species chase and capture butterflies during flight, not when they are landed (Carpenter 1939; Carpenter and Hale 1942; Chai 1986; Pinheiro and Cintra 2017). In-flight predation by birds likely accounts for the outcome of Experiment 1, since beak marks in Experiment 3 indicated scant predation attempts on both treatment and control butterflies landed on the forest floor. Given that treatment and control butterflies were manipulated exactly in the same way in both experiments (same wing area), external factors potentially altering their survival or likelihood of being recaptured should affect both groups equally. Thus, different recapture rates between treatment and control M. helenor can reasonably be attributed to the color used in each group, that is, altering or not the dynamic flashing pattern during flight.

The decrease in the recapture probability of Morpho helenor, when the flash was changed during flight, suggests that color manipulation might have resulted in a higher incidence of successful predation of treated butterflies in the air, which became more vulnerable visual targets to chasing predators. Our results provide the first experimental evidence, to our knowledge, supporting dynamic flash coloration as a protective mechanism in Morpho butterflies against predation. Although previous studies have suggested that variation between cryptic and conspicuous colors during movement can decrease the chance of a given prey being captured, the supporting evidence is based only on virtual experiments using artificial prey and humans as predators (Loeffler-Henry et al. 2018; Murali 2018; Bae et al. 2019; Murali and Kodandaramaiah 2020; Loeffler-Henry, Kang, and Sherratt 2021; Sherratt and Loeffler-Henry 2022; Silvasti et al. 2024). Our experimental study is unique by using real prey and real predators, in their natural habitat, to corroborate flash coloration as a defense mechanism that potentially decreases the predator's ability to track the butterfly during flight.

Equal survival between treatment and control butterflies in Experiment 2 is likely due to the removal of the blue band in treatment individuals, which became cryptic to visually hunting birds during flight in the forest understory. There are different forms of crypsis that evolved in butterflies in their adult stage. In background matching, detection by predators is avoided due to the similarity of the animal's appearance—whether by color, brightness, contrast, or pattern—with one or more types of background (Stevens and Merilaita 2009). Many Satyrinae butterflies, including some Morpho species, are brown on the dorsal and ventral wing surfaces and are considered cryptic during flight (Schwanwitsch 1948). Thus, by removing the bright blue color from the dorsal face of M. helenor, we made the color pattern of treated butterflies cryptically colored during flight (as reported for brown satyrines), which may explain the outcome of Experiment 2. Indeed, a uniform color pattern resembling the background can be advantageous for moving prey. Murali (2018) has shown that artificial prey with background matching pattern were less caught by humans than prey with white coloration when in motion, suggesting that crypsis may have some advantage over more conspicuous coloration for moving prey. Furthermore, other studies have demonstrated that targets exhibiting a uniform color that resembles the average background color can be difficult to capture (Stevens, Yule, and Ruxton 2008; Hughes, Troscianko, and Stevens 2014; Hughes, Magor-Elliott, and Stevens 2015), and can be just as effective at escaping from humans as dynamically changing color targets (Murali and Kodandaramaiah 2020).

Therefore, one may ask why M. helenor has not evolved cryptic coloration during flight, as many other Satyrinae. Indeed, the adaptive value of the iridescent blue color in Morpho butterflies, and the factors that might have shaped the evolution of this color pattern, have been asked for a long time. This blue color was already present in males of the lineages that first diverged in the group, Morpho marcus and Morpho eugenia, suggesting that blue iridescence was present in the ancestor of Morpho and this taxonomic character has been maintained in a large part of the group (Chazot et al. 2016; Debat et al. 2018). The evolution of coloration patterns is under various selective pressures, which are often highly interconnected (Caro, Sherratt, and Stevens 2016; Cuthill et al. 2017), such as intraspecific for communication, thermoregulation, and defense against predation (Caro, Sherratt, and Stevens 2016). For M. helenor, intraspecific communication appears to have played no important role in the evolution of the blue pattern, given that there is no marked sexual dimorphism in this species, and males do not have any kind of territorial behavior towards other males (Blandin and Morris 2007). The importance of wing color for thermoregulation in Morpho is unclear. Berthier (2010) claims that the iridescent blue and its high reflectivity do not seem to play such an important role in absorption capacity. More recently, Thomé, Richalot, and Berthier (2020) suggests that the structure of the wing scales is related to a phenomenon that helps thermoregulation. As far as we know, no work has assessed whether the scale structure and iridescence of M. helenor wings could have any advantage for thermoregulation. Our field experiments with M. helenor provide evidence that defense against predation via flash coloration during flight might have been an important factor shaping the evolution of the iridescent blue coloration in this species. Indeed, Le Roy et al. (2021) recently suggested that similar wing color patterns of sympatric Morpho species with erratic flight abilities may function as escape mimicry and discourage further predatory attacks. Further investigation integrating multiple selective forces should help clarify the evolution of the flashy, iridescent coloration pattern in Morpho butterflies.

For 160 years, naturalists (e.g., Bates 1863) have marveled at the flashing and intense blue color of Morpho butterflies, however, tests of adaptive explanations of flash coloration in these butterflies have hitherto been absent (Debat et al. 2018). Here we demonstrate experimentally that the flashy blue color of M. helenor is an effective defense mechanism against visually oriented predators, mostly birds, in Atlantic rainforest. We suggest that the dynamic flash coloration during flight makes it difficult for predators to chase and capture the butterflies in the shady forest understory. The phenomenon of dynamic flash coloration is known to occur in many Morpho species (Chazot et al. 2016), as well as in other butterfly genera, such as Doxocopa and Myscelia. Our experimental study is a first step to understand the adaptive value of such a widely distributed trait that combines color and behavior, and which is so commonly observed among insects in tropical and temperate environments.

Author Contributions

Aline Vieira-Silva: conceptualization, investigation, methodology, validation, formal analysis, data curation, visualization, writing – original draft, software. Gabriel B. Evora: formal analysis, investigation, writing – review and editing, software. André V. L. Freitas: conceptualization, methodology, visualization, writing – review and editing. Paulo S. Oliveira: conceptualization, data curation, funding acquisition, methodology, supervision, visualization, writing – review and editing, project administration, resources.

Ethics Statement

This study followed ethical standards and was conducted under permits issued by the Biodiversity Authorization and Information System (SISBIO, Brazil) with license numbers 10438-6 and 73086.

Conflicts of Interest

The authors declare no conflicts of interest.