Bumblebees compensate for the adverse effects of sidewind during visually guided landings.

Flying animals often encounter winds during visually guided landings. However, how winds affect their flight control strategy during landing is unknown. Here, we investigated how sidewind affects the landing performance and sensorimotor control of foraging bumblebees (Bombus terrestris). We trained bumblebees to forage in a wind tunnel, and used high-speed stereoscopic videography to record 19,421 landing maneuvers in six sidewind speeds (0 to 3.4 m s-1), which correspond to winds encountered in nature. Bumblebees landed less often in higher windspeeds, but the landing durations from free flight were not increased by wind. By testing how bumblebees adjusted their landing control to compensate for adverse effects of sidewind on landing, we showed that the landing strategy in sidewind resembled that in still air, but with important adaptations. Bumblebees landing in a sidewind tended to drift downwind, which they controlled for by performing more hover maneuvers. Surprisingly, the increased hover prevalence did not increase the duration of free-flight landing maneuvers, as these bumblebees flew faster towards the landing platform outside the hover phases. Hence, by alternating these two flight modes along their flight path, free-flying bumblebees negated the adverse effects of high windspeeds on landing duration. Using control theory, we hypothesize that bumblebees achieve this by integrating a combination of direct aerodynamic feedback and a wind-mediated mechanosensory feedback control, with their vision-based sensorimotor control loop. The revealed landing strategy may be commonly used by insects landing in windy conditions, and may inspire the development of landing control strategies onboard autonomously flying robots.

Predicting the effects of spatiotemporal modifications of muscle activation on the tentacle extension in squid.

Squid use eight arms and two slender tentacles to capture prey. The muscular stalks of the tentacles are elongated approximately 80% in 20-40 ms towards the prey, which is adhered to the terminal clubs by arrays of suckers. Using a previously developed forward dynamics model of the extension of the tentacles of the squid Doryteuthis pealeii (formerly Loligo pealeii), we predict how spatial muscle-activation patterns result in a distribution of muscular power, muscle work, and kinetic and elastic energy along the tentacle. The simulated peak extension speed of the tentacles is remarkably insensitive to delays of activation along the stalk, as well as to random variations in the activation onset. A delay along the tentacle of 50% of the extension time has only a small effect on the peak extension velocity of the tentacle compared with a zero-delay pattern. A slight delay of the distal portion relative to the proximal has a small positive effect on peak extension velocity, whereas negative delays (delay reversed along stalk) always reduce extension performance. In addition, tentacular extension is relatively insensitive to superimposed random variations in the prescribed delays along the stalk. This holds in particular for small positive delays that are similar to delays predicted from measured axonal diameters of motor neurons. This robustness against variation in the activation distribution reduces the accuracy requirements of the neuronal control and is likely due to the non-linear mechanical properties of the muscular tissue in the tentacle.

Cost of Transport of Undulating Fin Propulsion.

Autonomous robots are used to inspect, repair and maintain underwater assets. These tasks require energy-efficient robots, including efficient movement to extend available operational time. To examine the suitability of a propulsion system based on undulating fins, we built two robots with one and two fins, respectively, and conducted a parametric study for combinations of frequency, amplitude, wavenumber and fin shapes in free-swimming experiments, measuring steady-state swimming speed, power consumption and cost of transport. The following trends emerged for both robots. Swimming speed was more strongly affected by frequency than amplitude across the examined wavenumbers and fin heights. Power consumption was sensitive to frequency at low wavenumbers, and increasingly sensitive to amplitude at high wavenumbers. This increasing sensitivity of amplitude was more pronounced in tall rather than short fins. Cost of transport showed a complex relation with fin size and kinematics and changed drastically across the mapped parameter space. At equal fin kinematics as the single-finned robot, the double-finned robot swam slightly faster (>10%) with slightly lower power consumption (<20%) and cost of transport (<40%). Overall, the robots perform similarly to finned biological swimmers and other bio-inspired robots, but do not outperform robots with conventional propulsion systems.

Multiple paternity in superfetatious live-bearing fishes.

Superfetation, the ability to carry several overlapping broods at different developmental stages, has evolved independently multiple times within the live-bearing fish family Poeciliidae. Even though superfetation is widespread among poeciliids, its evolutionary advantages remain unclear. Theory predicts that superfetation should increase polyandry by increasing the probability that temporally overlapping broods are fertilized by different fathers. Here, we test this key prediction in two poeciliid species that each carry two temporally overlapping broods: Poeciliopsis retropinna and P. turrubarensis. We collected 25 females per species from freshwater streams in South-Eastern Costa Rica and assessed multiple paternity by genotyping all their embryos (420 embryos for P. retropinna; 788 embryos for P. turrubarensis) using existing and newly developed microsatellite markers. We observed a high frequency of unique sires in the simultaneous, temporally overlapping broods in P. retropinna (in 56% of the pregnant females) and P. turrubarensis (79%). We found that the mean number of sires within females was higher than the number of sires within the separate broods (2.92 sires within mothers vs. 2.36 within separate broods in P. retropinna; and 3.40 vs 2.56 in P. turrubarensis). We further observed that there were significant differences in the proportion of offspring sired by each male in 42% of pregnant female P. retropinna and 65% of female P. turrubarensis; however, this significance applied to only 9% and 46% of the individual broods in P. retropinna and P. turrubarensis, respectively, suggesting that the unequal reproductive success of sires (i.e. reproductive skew) mostly originated from differences in paternal contribution between, rather than within broods. Together, these findings tentatively suggest that superfetation may promote polyandry and reproductive skew in live-bearing fishes.

Bumblebees land rapidly by intermittently accelerating and decelerating toward the surface during visually guided landings.

Many flying animals parse visual information to control their landing, whereby they can decelerate smoothly by flying at a constant radial optic expansion rate. Here, we studied how bumblebees (Bombus terrestris) use optic expansion information to control their landing, by analyzing 10,005 landing maneuvers on vertical platforms with various optic information, and at three dim light conditions. We showed that bumblebees both decelerate and accelerate during these landings. Bumblebees decelerate by flying at a constant optic expansion rate, but they mostly accelerate toward the surface each time they switched to a new, often higher, optic expansion rate set-point. These transient acceleration phases allow bumblebees to increase their approach speed, and thereby land rapidly and robustly, even in dim twilight conditions. This helps explain why bumblebees are such robust foragers in challenging environmental conditions. The here-proposed sensorimotor landing control system can serve as bio-inspiration for landing control in unmanned aerial vehicles.

Diurnal and nocturnal mosquitoes escape looming threats using distinct flight strategies.

Flying insects have evolved the ability to evade looming objects, such as predators and swatting hands. This is particularly relevant for blood-feeding insects, such as mosquitoes that routinely need to evade the defensive actions of their blood hosts. To minimize the chance of being swatted, a mosquito can use two distinct strategies-continuously exhibiting an unpredictable flight path or maximizing its escape maneuverability. We studied how baseline flight unpredictability and escape maneuverability affect the escape performance of day-active and night-active mosquitoes (Aedes aegypti and Anopheles coluzzii, respectively). We used a multi-camera high-speed videography system to track how freely flying mosquitoes respond to an event-triggered rapidly approaching mechanical swatter, in four different light conditions ranging from pitch darkness to overcast daylight. Results show that both species exhibit enhanced escape performance in their natural blood-feeding light condition (daylight for Aedes and dark for Anopheles). To achieve this, they show strikingly different behaviors. The enhanced escape performance of Anopheles at night is explained by their increased baseline unpredictable erratic flight behavior, whereas the increased escape performance of Aedes in overcast daylight is due to their enhanced escape maneuvers. This shows that both day and night-active mosquitoes modify their flight behavior in response to light intensity such that their escape performance is maximum in their natural blood-feeding light conditions, when these defensive actions by their blood hosts occur most. Because Aedes and Anopheles mosquitoes are major vectors of several deadly human diseases, this knowledge can be used to optimize vector control methods for these specific species.

Maternal food restriction during pregnancy affects offspring development and swimming performance in a placental live-bearing fish.

How pregnant mothers allocate limited resources to different biological functions such as maintenance, somatic growth, and reproduction can have profound implications for early life development and survival of offspring. Here, we examined the effects of maternal food restriction during pregnancy on offspring in the matrotrophic (i.e. mother-nourishment throughout gestation) live-bearing fish species Phalloptychus januarius (Poeciliidae). We fed pregnant females with either low or high food levels for 6 weeks and quantified the consequences for offspring size and body fat at birth and 1 week after birth. We further measured fast-start escape performance of offspring at birth, as well as swimming kinematics during prey capture at 0, 2 and 7 days after birth. We found that the length of maternal food restriction during pregnancy negatively affected offspring dry mass and lean dry mass at birth, as well as body fat gain during the first week after birth. Moreover, it impacted the locomotor performance of offspring during prey capture at birth and during the first week after birth. We did not observe an effect of food restriction on fast-start escape performance of offspring. Our study suggests that matrotrophic poeciliid fish are maladapted to unpredictably fluctuating resource environments, because sudden reductions in maternal food availability during pregnancy result in smaller offspring with slower postnatal body fat gain and an inhibition of postnatal improving swimming skills during feeding, potentially leading to lower competitive abilities after birth.

Fishes regulate tail-beat kinematics to minimize speed-specific cost of transport.

Energetic expenditure is an important factor in animal locomotion. Here we test the hypothesis that fishes control tail-beat kinematics to optimize energetic expenditure during undulatory swimming. We focus on two energetic indices used in swimming hydrodynamics, cost of transport and Froude efficiency. To rule out one index in favour of another, we use computational-fluid dynamics models to compare experimentally observed fish kinematics with predicted performance landscapes and identify energy-optimized kinematics for a carangiform swimmer, an anguilliform swimmer and larval fishes. By locating the areas in the predicted performance landscapes that are occupied by actual fishes, we found that fishes use combinations of tail-beat frequency and amplitude that minimize cost of transport. This energy-optimizing strategy also explains why fishes increase frequency rather than amplitude to swim faster, and why fishes swim within a narrow range of Strouhal numbers. By quantifying how undulatory-wave kinematics affect thrust, drag, and power, we explain why amplitude and frequency are not equivalent in speed control, and why Froude efficiency is not a reliable energetic indicator. These insights may inspire future research in aquatic organisms and bioinspired robotics using undulatory propulsion.

Bumblebees land rapidly and robustly using a sophisticated modular flight control strategy.

When approaching a landing surface, many flying animals use visual feedback to control their landing. Here, we studied how foraging bumblebees (Bombus terrestris) use radial optic expansion cues to control in-flight decelerations during landing. By analyzing the flight dynamics of 4,672 landing maneuvers, we showed that landing bumblebees exhibit a series of deceleration bouts, unlike landing honeybees that continuously decelerate. During each bout, the bumblebee keeps its relative rate of optical expansion constant, and from one bout to the next, the bumblebee tends to shift to a higher, constant relative rate of expansion. This modular landing strategy is relatively fast compared to the strategy described for honeybees and results in approach dynamics that is strikingly similar to that of pigeons and hummingbirds. The here discovered modular landing strategy of bumblebees helps explaining why these important pollinators in nature and horticulture can forage effectively in challenging conditions; moreover, it has potential for bio-inspired landing strategies in flying robots.

Lure, retain, and catch malaria mosquitoes. How heat and humidity improve odour-baited trap performance.

BACKGROUND: When seeking a human for a blood meal, mosquitoes use several cues to detect and find their hosts. From this knowledge, counter-flow odour-baited traps have been developed that use a combination of CO2, human-mimicking odour, visual cues and circulating airflow to attract and capture mosquitoes. Initially developed for monitoring, these traps are now also being considered as promising vector control tools. The traps are attractive to host-seeking mosquitoes, but their capture efficiency is low. It has been hypothesized that the lack of short-range host cues, such as heat and increased local humidity, often prevent mosquitoes from getting close enough to get caught; this lack might even trigger avoidance manoeuvres near the capture region. METHODS: This study investigated how close-range host cues affect the flight behaviour of Anopheles female malaria mosquitoes around odour-baited traps, and how this affects trap capture performance. For this, a novel counter-flow odour-baited trap was developed, the M-Tego. In addition to the usual CO2 and odour-blend, this trap can provide the short-range host cues, heat and humidity. Systematically adding or removing these two cues tested how this affected the trap capture percentages and flight behaviour. First, capture percentages of the M-Tego with and without short-range host cues to the BG-Suna trap were compared, in both laboratory and semi-field testing. Then, machine-vision techniques were used to track the three-dimensional flight movements of mosquitoes around the M-Tego. RESULTS: With heat and humidity present, the M-Tego captured significantly more mosquitoes as capture percentages almost doubled. Comparing the flight behaviour around the M-Tego with variable close-range host cues showed that when these cues were present, flying mosquitoes were more attracted to the trap and spent more time there. In addition, the M-Tego was found to have a better capture mechanism than the BG-Suna, most likely because it does not elicit previously observed upward avoiding manoeuvres. CONCLUSIONS: Results suggest that adding heat and humidity to an odour-baited trap lures more mosquitoes close to the trap and retains them there longer, resulting in higher capture performance. These findings support the development of control tools for fighting mosquito-borne diseases such as malaria.

Experimental-numerical method for calculating bending moments in swimming fish shows that fish larvae control undulatory swimming with simple actuation.

Most fish swim with body undulations that result from fluid-structure interactions between the fish's internal tissues and the surrounding water. Gaining insight into these complex fluid-structure interactions is essential to understand how fish swim. To this end, we developed a dedicated experimental-numerical inverse dynamics approach to calculate the lateral bending moment distributions for a large-amplitude undulatory swimmer that moves freely in three-dimensional space. We combined automated motion tracking from multiple synchronised high-speed video sequences, computation of fluid dynamic stresses on the swimmer's body from computational fluid dynamics, and bending moment calculations using these stresses as input for a novel beam model of the body. The bending moment, which represent the system's net actuation, varies over time and along the fish's central axis due to muscle actions, passive tissues, inertia, and fluid dynamics. Our three-dimensional analysis of 113 swimming events of zebrafish larvae ranging in age from 3 to 12 days after fertilisation shows that these bending moment patterns are not only relatively simple but also strikingly similar throughout early development and from fast starts to periodic swimming. This suggests that fish larvae may produce and adjust swimming movements relatively simply, yet effectively, while restructuring their neuromuscular control system throughout their rapid development.

Bladderworts, the smallest known suction feeders, generate inertia-dominated flows to capture prey.

Aquatic bladderworts (Utricularia gibba and U. australis) capture zooplankton in mechanically triggered underwater traps. With characteristic dimensions less than 1 mm, the trapping structures are among the smallest known to capture prey by suction, a mechanism that is not effective in the creeping-flow regime where viscous forces prevent the generation of fast and energy-efficient suction flows. To understand what makes suction feeding possible on the small scale of bladderwort traps, we characterised their suction flows experimentally (using particle image velocimetry) and mathematically (using computational fluid dynamics and analytical mathematical models). We show that bladderwort traps avoid the adverse effects of creeping flow by generating strong, fast-onset suction pressures. Our findings suggest that traps use three morphological adaptations: the trap walls' fast release of elastic energy ensures strong and constant suction pressure; the trap door's fast opening ensures effectively instantaneous onset of suction; the short channel leading into the trap ensures undeveloped flow, which maintains a wide effective channel diameter. Bladderwort traps generate much stronger suction flows than larval fish with similar gape sizes because of the traps' considerably stronger suction pressures. However, bladderworts' ability to generate strong suction flows comes at considerable energetic expense.

The ovipositor actuation mechanism of a parasitic wasp and its functional implications.

Parasitic wasps use specialized needle-like structures, ovipositors, to drill into substrates to reach hidden hosts. The external ovipositor (terebra) consists of three interconnected, sliding elements (valvulae), which are moved reciprocally during insertion. This presumably reduces the required pushing force on the terebra and limits the risk of damage whilst probing. Although this is an important mechanism, it is still not completely understood how the actuation of the valvulae is achieved, and it has only been studied with the ovipositor in rest position. Additionally, very little is known about the magnitude of the forces generated during probing. We used synchrotron X-ray microtomography to reconstruct the actuation mechanism of the parasitic wasp Diachasmimorpha longicaudata (Braconidae) in four distinct phases of the probing cycle. We show that only the paired first valvulae of the terebra move independently, while the second valvula moves with the metasoma ('abdomen'). The first valvula movements are initiated by rotation of one chitin plate (first valvifer) with respect to another such plate (second valvifer). This is achieved indirectly by muscles connecting the non-rotating second valvifer and the abdominal ninth tergite. Contrary to previous reports, we found muscle fibres running inside the terebra, although their function remains unclear. The estimated maximal forces that can be exerted by the first valvulae are small (protraction 1.19 mN and retraction 0.874 mN), which reduces the risk of buckling, but are sufficient for successful probing. The small net forces of the valvulae on the substrate may still lead to buckling of the terebra; we show that the sheaths surrounding the valvulae prevent this by effectively increasing the diameter and second moment of area of the terebra. Our findings improve the comprehension of hymenopteran probing mechanisms, the function of the associated muscles, and the forces and damage-limiting mechanism that are involved in drilling a slender terebra into a substrate.

Bumblebees land remarkably well in red-blue greenhouse LED light conditions.

Red-blue emitting LEDs have recently been introduced in greenhouses to optimise plant growth. However, this spectrum may negatively affect the performance of bumblebees used for pollination, because the visual system of bumblebees is more sensitive to green light than to red-blue light. We used high-speed stereoscopic videography to three-dimensionally track and compare landing manoeuvres of Bombus terrestris bumblebees in red-blue light and in regular, broad-spectrum white light. In both conditions, the landing approaches were interspersed by one or several hover phases, followed by leg extension and touchdown. The time between leg extension and touchdown was 25% (0.05 s) longer in red-blue light than in white light, caused by a more tortuous flight path in red-blue light. However, the total landing duration, specified as the time between the first hover phase and touchdown, did not differ between the light conditions. This suggests that the negative effects of red-blue light on the landing manoeuvre are confined to the final phase of the landing.This article has an associated First Person interview with the first author of the paper.

Design of Tree-Frog-Inspired Adhesives.

The adhesive toe pads of tree frogs have inspired the design of various so-called 'smooth' synthetic adhesives for wet environments. However, these adhesives do not reach the attachment performance of their biological models in terms of contact formation, maintenance of attachment, and detachment. In tree frogs, attachment is facilitated by an interconnected ensemble of superficial and internal morphological components, which together form a functional unit. To help bridging the gap between biological and bioinspired adhesives, in this review, we (1) provide an overview of the functional components of tree frog toe pads, (2) investigate which of these components (and attachment mechanisms implemented therein) have already been transferred into synthetic adhesives, and (3) highlight functional analogies between existing synthetic adhesives and tree frogs regarding the fundamental mechanisms of attachment. We found that most existing tree-frog-inspired adhesives mimic the micropatterned surface of the ventral epidermis of frog pads. Geometrical and material properties differ between these synthetic adhesives and their biological model, which indicates similarity in appearance rather than function. Important internal functional components such as fiber-reinforcement and muscle fibers for attachment control have not been considered in the design of tree-frog-inspired adhesives. Experimental work on tree-frog-inspired adhesives suggests that the micropatterning of adhesives with low-aspect-ratio pillars enables crack arresting and the drainage of interstitial liquids, which both facilitate the generation of van der Waals forces. Our analysis of experimental work on tree-frog-inspired adhesives indicates that interstitial liquids such as the mucus secreted by tree frogs play a role in detachment. Based on these findings, we provide suggestions for the future design of biomimetic adhesives. Specifically, we propose to implement internal fiber-reinforcements inspired by the fibrous structures in frog pads to create mechanically reinforced soft adhesives for high-load applications. Contractile components may stimulate the design of actuated synthetic adhesives with fine-tunable control of attachment strength. An integrative approach is needed for the design of tree-frog-inspired adhesives that are functionally analogous with their biological paradigm.

Malaria mosquitoes use leg push-off forces to control body pitch during take-off.

Escaping from a blood host with freshly acquired nutrition for her eggs is one of the most critical actions in the life of a female malaria mosquito. During this take-off, she has to carry a large payload, up to three times her body weight, while avoiding tactile detection by the host. What separates the malaria mosquito from most other insects is that the mosquito pushes off gently with its legs while producing aerodynamic forces with its wings. Apart from generating the required forces, the malaria mosquito has to produce the correct torques to pitch-up during take-off. Furthermore, the fed mosquito has to alter the direction of its aerodynamic force vector to compensate for the higher body pitch angle due to its heavier abdomen. Whether the mosquito generates these torques and redirection of the forces with its wings or legs remains unknown. By combining rigid-body inverse dynamics analyses with computational fluid dynamics simulations, we show that mosquitoes use leg push-off to control pitch torques and that the adaption of the aerodynamic force direction is synchronized with modulations in force magnitude. These results suggest that during the push-off phase of a take-off, mosquitoes use their flight apparatus primarily as a motor system and they use leg push-off forces for control.

Superfetation reduces the negative effects of pregnancy on the fast-start escape performance in live-bearing fish.

Superfetation, the ability to simultaneously carry multiple litters of different developmental stages in utero, is a reproductive strategy that evolved repeatedly in viviparous animal lineages. The evolution of superfetation is hypothesized to reduce the reproductive burden and, consequently, improve the locomotor performance of the female during pregnancy. Here, we apply new computer-vision-based techniques to study changes in body shape and three-dimensional fast-start escape performance during pregnancy in three live-bearing fishes (family Poeciliidae) that exhibit different levels of superfetation. We found that superfetation correlates with a reduced abdominal distension and a more slender female body shape just before parturition. We further found that body slenderness positively correlates with maximal speeds, curvature amplitude and curvature rate, implying that superfetation improves the fast-start escape performance. Collectively, our study suggests that superfetation may have evolved in performance-demanding (e.g. high flow or high predation) environments to reduce the locomotor cost of pregnancy.

Reorientation and propulsion in fast-starting zebrafish larvae: an inverse dynamics analysis.

Most fish species use fast starts to escape from predators. Zebrafish larvae perform effective fast starts immediately after hatching. They use a C-start, where the body curls into a C-shape, and then unfolds to accelerate. These escape responses need to fulfil a number of functional demands, under the constraints of the fluid environment and the larva's body shape. Primarily, the larvae need to generate sufficient escape speed in a wide range of possible directions, in a short-enough time. In this study, we examined how the larvae meet these demands. We filmed fast starts of zebrafish larvae with a unique five-camera setup with high spatiotemporal resolution. From these videos, we reconstructed the 3D swimming motion with an automated method and from these data calculated resultant hydrodynamic forces and, for the first time, 3D torques. We show that zebrafish larvae reorient mostly in the first stage of the start by producing a strong yaw torque, often without using the pectoral fins. This reorientation is expressed as the body angle, a measure that represents the rotation of the complete body, rather than the commonly used head angle. The fish accelerates its centre of mass mostly in stage 2 by generating a considerable force peak while the fish 'unfolds'. The escape direction of the fish correlates strongly with the amount of body curvature in stage 1, while the escape speed correlates strongly with the duration of the start. This may allow the fish to independently control the direction and speed of the escape.

A chordwise offset of the wing-pitch axis enhances rotational aerodynamic forces on insect wings: a numerical study.

Most flying animals produce aerodynamic forces by flapping their wings back and forth with a complex wingbeat pattern. The fluid dynamics that underlies this motion has been divided into separate aerodynamic mechanisms of which rotational lift, that results from fast wing pitch rotations, is particularly important for flight control and manoeuvrability. This rotational force mechanism has been modelled using Kutta-Joukowski theory, which combines the forward stroke motion of the wing with the fast pitch motion to compute forces. Recent studies, however, suggest that hovering insects can produce rotational forces at stroke reversal, without a forward motion of the wing. We have conducted a broad numerical parametric study over a range of wing morphologies and wing kinematics to show that rotational force production depends on two mechanisms: (i) conventional Kutta-Joukowski-based rotational forces and (ii) a rotational force mechanism that enables insects with an offset of the pitch axis relative to the wing's chordwise symmetry axis to generate rotational forces in the absence of forward wing motion. Because flying animals produce control actions frequently near stroke reversal, this pitch-axis-offset dependent aerodynamic mechanism may be particularly important for understanding control and manoeuvrability in natural flyers.

Comparative and functional analysis of the digital mucus glands and secretions of tree frogs.

BACKGROUND: Mucus and mucus glands are important features of the amphibian cutis. In tree frogs, the mucus glands and their secretions are crucial components of the adhesive digital pads of these animals. Despite a variety of hypothesised functions of these components in tree frog attachment, the functional morphology of the digital mucus glands and the chemistry of the digital mucus are barely known. Here, we use an interdisciplinary comparative approach to analyse these components, and discuss their roles in tree frog attachment. RESULTS: Using synchrotron micro-computer-tomography, we discovered in the arboreal frog Hyla cinerea that the ventral digital mucus glands differ in their morphology from regular anuran mucus glands and form a subdermal gland cluster. We show the presence of this gland cluster also in several other-not exclusively arboreal-anuran families. Using cryo-histochemistry as well as infrared and sum frequency generation spectroscopy on the mucus of two arboreal (H. cinerea and Osteopilus septentrionalis) and of two terrestrial, non-climbing frog species (Pyxicephalus adspersus and Ceratophrys cranwelli), we find neutral and acidic polysaccharides, and indications for proteinaceous and lipid-like mucus components. The mucus chemistry varies only little between dorsal and ventral digital mucus in H. cinerea, ventral digital and abdominal mucus in H. cinerea and O. septentrionalis, and between the ventral abdominal mucus of all four studied species. CONCLUSIONS: The presence of a digital mucus gland cluster in various anuran families, as well as the absence of differences in the mucus chemistry between arboreal and non-arboreal frog species indicate an adaptation towards generic functional requirements as well as to attachment-related requirements. Overall, this study contributes to the understanding of the role of glands and their secretions in tree frog attachment and in bioadhesion in general, as well as the evolution of anurans.