Evolutionary trade-offs between testes size and parenting in Neotropical glassfrogs.

In males, large testes size signifies high sperm production and is commonly linked to heightened sperm competition levels. It may also evolve as a response to an elevated risk of sperm depletion due to multiple mating or large clutch sizes. Conversely, weapons, mate or clutch guarding may allow individuals to monopolize mating events and preclude sperm competition, thereby reducing the selection of large testes. Herein, we examined how paternal care, sexual size dimorphism (SSD), weaponry and female fecundity are linked to testes size in glassfrogs. We found that paternal care was associated with a reduction in relative testes size, suggesting an evolutionary trade-off between testes size and parenting. Although females were slightly larger than males and species with paternal care tended to have larger clutches, there was no significant relationship between SSD, clutch size and relative testes size. These findings suggest that the evolution of testes size in glassfrogs is influenced by sperm competition risk, rather than sperm depletion risk. We infer that clutch guarding precludes the risk of fertilization by other males and consequently diminishes selective pressure for larger testes. Our study highlights the prominent role of paternal care in the evolution of testes size in species with external fertilization.

Water-borne testosterone levels predict exploratory tendency in male poison frogs.

Hormones play a fundamental role in mediating social behaviors of animals. However, it is less well understood to what extent behavioral variation between individuals can be attributed to variation in underlying hormonal profiles. The goal of the present study was to infer if individual androgen levels, and/or the modulation thereof, can explain among-individual variation in aggressiveness, boldness and exploration. We used as a model the dart-poison frog Allobates femoralis and took repeated non-invasive water-borne hormonal samples of individual males before (baseline) and after (experimental) a series of behavioral tests for assessing aggression, boldness, and exploratory tendency. Our results show that androgen levels in A. femoralis are quite stable across the reproductive season. Repeatability in wbT baseline levels was high, while time of day, age of the frog, and trial order did not show any significant impact on measured wbT levels. In general, experimental wbT levels after behavioral tests were lower compared to the respective baseline levels. However, we identified two different patterns with regard to androgen modulation in response to behavioral testing: individuals with low baseline wbT tended to have increased wbT levels after the behavioral testing, while individuals with comparatively high baseline wbT levels rather showed a decrease in hormonal levels after testing. Our results also suggest that baseline wbT levels are linked to the personality trait exploration, and that androgen modulation is linked to boldness in A. femoralis males. These results show that differences in hormonal profiles and/or hormonal modulation in response to social challenges can indeed explain among-individual differences in behavioral traits.

Egg burying behaviour in Pristimantis highlights the link between direct development and specialised parental care.

One of the most extreme adaptations to terrestriality in anurans is direct development, where eggs from terrestrial clutches entirely circumvent an aquatic tadpole stage and directly develop into small froglets. We here report the first case of egg-burying behaviour in a neotropical direct-developing frog with subsequent short-term maternal care. An amplectant pair of Pristimantis chocoensis was found at the Reserva Canandé in Esmeraldas, Ecuador, and we recorded oviposition and the later rotation and active burying of the clutch by the female. Both parents remained close to the nest area the following day. This rare observation sheds light on the complex but often cryptic reproductive behaviours of terrestrial amphibians and suggests that the evolution of direct development has selected for highly specialized forms of parental care.

Una de las adaptaciones más extremas a la terrestreidad en los anuros es el desarrollo directo, donde los huevos de puestas terrestres evitan por completo la etapa de renacuajo acuático y se desarrollan directamente en versiones miniatura de los adultos. Aquí informamos el primer caso de comportamiento de enterrar huevos en una rana neotropical de desarrollo directo con posterior cuidado materno a corto plazo. Una pareja amplectante de Pristimantis chocoensis fue encontrada en la Reserva Canandé en Esmeraldas­Ecuador, y registramos la oviposición y la posterior rotación y enterramiento activo de la puesta por parte de la hembra. Ambos padres permanecieron cerca del área del nido durante el día siguiente. Esta rara observación arroja luz sobre los complejos, pero a menudo crípticos, comportamientos reproductivos de los anfibios terrestres y sugiere que la evolución del desarrollo directo ha seleccionado formas altamente especializadas de cuidado parental.

Personality traits differentially affect components of reproductive success in a Neotropical poison frog.

Individual reproductive success has several components, including the acquisition of mating partners, offspring production, and offspring survival until adulthood. While the effects of certain personality traits-such as boldness or aggressiveness-on single components of reproductive success are well studied, we know little about the composite and multifaceted effects behavioural traits can have on all the aspects of reproductive success. Behavioural traits positively linked to one component of reproductive success might not be beneficial for other components, and these effects may differ between sexes. We investigated the influence of boldness, aggressiveness, and exploration on the number of mating partners, mating events, and offspring surviving until adulthood in males and females of the Neotropical poison frog Allobates femoralis. Behavioural traits had different-even opposite-effects on distinct components of reproductive success in both males and females. For example, males who displayed high levels of aggressiveness and exploration (or low levels of aggressiveness and exploration) managed to attract high number of mating partners, while males with low levels of boldness, low levels of aggressiveness, and high levels of exploration had the most offspring surviving until adulthood. Our results therefore suggest correlational selection favouring particular combinations of behavioural traits.

Regardless of personality, males show similar levels of plasticity in territory defense in a Neotropical poison frog.

Animal personality traits are sometimes linked to an individual's degree of plasticity, with certain personality types being more plastic than others. In territorial species, consistently high levels of aggression might increase the risk of harmful fights, while consistently low aggression might lead to the loss of a territory. Consequently, reacting plastically with an appropriate territorial response should be beneficial to avoid these risks. An integrative investigation of both personality traits and plasticity can help us better understand the dynamics of aggressive interactions during male-male competition. Here, we used a free-ranging Neotropical poison frog population to investigate the role of plasticity in male territorial aggression towards intruders. We conducted repeated standardized territorial intrusion experiments mimicking frogs of different body sizes via playback calls with different peak frequencies. We found individual repeatability for the latency to reach and approach a simulated intruder and observed that both aggressive and less aggressive males decreased their level of aggression towards big intruders. However, our results do not support a correlation between personality and plasticity in the context of male territory defense during the breeding season. We discuss how such a correlation between personality and plasticity might be conditional on the costs and benefits across contexts.

Complement protein C3a enhances adaptive immune responses towards FVIII products.

The most serious complication in the treatment of hemophilia A (HA) is the development of factor (F)VIII inhibitors or antidrug antibodies (ADA) occurring in 25-35% of patients with severe HA. The immunological mechanisms underlying the development of ADA against FVIII products have not been completely understood yet. Immunological danger signals associated with events such as infection or surgery have been suggested to play a critical role. In previous studies, we demonstrated that plasma-derived (pd)FVIII but not recombinant (r)FVIII can activate human monocyte-derived dendritic cells (DC) in a danger signal-dependent manner, which subsequently mediate the proliferation of autologous CD4+ T cells. In this study, we investigated the ability of plasma components, naturally present in pdFVIII products, to mediate T-cell responses. In fact, we show that addition of plasma to rFVIII plus lipopolysaccharide (LPS)-stimulated DC induces proliferation of autologous CD4+ T cells. Interestingly, although DC pulsed with LPS plus plasma induce T-cell proliferation upon co-culture, the addition of FVIII significantly increases the number of proliferating as well as FVIII-specific CD4+ T cells. Total proliferating CD4+ T cells and FVIII-specific subsets were identified mainly as central memory T cells. Experiments using blocking antibodies and receptor antagonists revealed that the complement proteins C3a and, to a lesser extent, C5a are critically involved in these LPS-mediated T-cell responses. Collectively, our results indicate that complement proteins are potent drivers of T-cell responses to FVIII. Data presented provide a model how event-related substitution of FVIII in HA patients might contribute to inhibitor development.

Geckos differentiate self from other using both skin and faecal chemicals: evidence towards self-recognition?

Self-recognition is the ability to recognise stimuli originating from oneself. Humans and some non-human animals show evidence of true visual self-recognition in the mirror test. They use their reflection to inspect themselves and to remove a mark that is only visible in the mirror. Not all animals, however, rely primarily on vision. In lizards, chemical cues are crucial in social interactions, and therefore, lizards would benefit from a chemical self-other distinction. Here, we test the tokay gecko (Gekko gecko), a social species, on their ability to discriminate their own skin and faecal chemicals from those of same-sex, unfamiliar conspecifics. We predicted that individuals would show more self-directed behaviour when confronted with the chemicals from unfamiliar individuals within their home enclosure as a sign of the need for increased comparison. Geckos showed higher self-directed responses towards chemicals from unfamiliar individuals compared to self-produced chemicals and a water control. Furthermore, scat and skin chemicals (regardless of origin) elicited similar but stronger responses than peppermint oil pointing towards a possible social function of scat piles. Although further tests and controls are needed, our study provides evidence towards chemical self-recognition and for a social function of scat piling in tokay geckos.

Fear of the new? Geckos hesitate to attack novel prey, feed near objects and enter a novel space.

Neophobia, the fear of novelty, is an ecologically important response which enables animals to avoid potentially harmful situations. Neophobia is a cognitive process by which individuals distinguish novelty from familiarity. In this study, we aimed to quantify this cognitive process in captive tokay geckos (Gekko gecko) across three contexts: when encountering novel prey, foraging near novel objects and entering a novel space. We also investigated individual consistency across trials using different novel stimuli, and correlation of individual responses across the three contexts. We found that geckos hesitate to attack novel prey and prey close to objects (familiar and novel). Geckos hesitated the most when entering novel space. Repeatability of behaviour within and across contexts was low (R = 0.101-0.190) indicating that neophobia might not be expressed similarly across contexts. The strength of a neophobic response can indicate how anxious or curious an individual is. This test has great potential to help answer questions about how captivity, enrichment, rearing environment and cognition affect fear responses in different contexts in lizards. By studying reptiles, we can better understand the universality of what is known about the causes leading to difference in neophobia across individuals and species.

Contrasting parental roles shape sex differences in poison frog space use but not navigational performance.

Sex differences in vertebrate spatial abilities are typically interpreted under the adaptive specialization hypothesis, which posits that male reproductive success is linked to larger home ranges and better navigational skills. The androgen spillover hypothesis counters that enhanced male spatial performance may be a byproduct of higher androgen levels. Animal groups that include species where females are expected to outperform males based on life-history traits are key for disentangling these hypotheses. We investigated the association between sex differences in reproductive strategies, spatial behavior, and androgen levels in three species of poison frogs. We tracked individuals in natural environments to show that contrasting parental sex roles shape sex differences in space use, where the sex performing parental duties shows wider-ranging movements. We then translocated frogs from their home areas to test their navigational performance and found that the caring sex outperformed the non-caring sex only in one out of three species. In addition, males across species displayed more explorative behavior than females and androgen levels correlated with explorative behavior and homing accuracy. Overall, we reveal that poison frog reproductive strategies shape movement patterns but not necessarily navigational performance. Together this work suggests that prevailing adaptive hypotheses provide an incomplete explanation of sex differences in spatial abilities.

Lizards lack speed-accuracy trade-offs in a quantitative foraging task when unable to sample the reward.

To make decisions, animals gather information from the environment in order to avoid costs (e.g., reduced survival) and increase benefits (e.g., foraging success). When time is limited or information is insufficient, most animals face a speed-accuracy trade-off (SAT) - they have to balance the benefits of making quick decisions against the costs of inaccurate decisions. Here, we investigated the relationship between decision accuracy and decision speed in gidgee skinks (Egernia stokesii) performing a food-based spontaneous quantity discrimination task. Rather than a SAT we found a speed-accuracy alignment; lizards made decisions that were fast and accurate, rather than inaccurate. Furthermore, we found only within-, but no between-individual differences in decision making indicating behavioural plasticity in the absence of individual decision styles. Finally, latency to choice was highly repeatable, more so than choice accuracy. Previous work has shown that learning, the costs of a bad decision and task difficulty frequently result in SATs. The lack of a SAT in our lizards might be a direct consequence of our simple testing methodology which prevented learning by not allowing lizards to consume the chosen quantity. To fully understand how SATs develop, different methodologies that control the costs and benefits of decisions should be compared.

Androgen responsiveness to simulated territorial intrusions in Allobates femoralis males: Evidence supporting the challenge hypothesis in a territorial frog.

Territoriality has been widely described across many animal taxa, where the acquisition and defence of a territory are critical for the fitness of an individual. Extensive evidence suggests that androgens are involved in the modulation of territorial behaviours in male vertebrates. Short-term increase of androgen following a territorial encounter appears to favour the outcome of a challenge. The "Challenge Hypothesis" proposed by Wingfield and colleagues outlines the existence of a positive feedback relationship between androgen and social challenges (e.g., territorial intrusions) in male vertebrates. Here we tested the challenge hypothesis in the highly territorial poison frog, Allobates femoralis, in its natural habitat by exposing males to simulated territorial intrusions in the form of acoustic playbacks. We quantified repeatedly androgen concentrations of individual males via a non-invasive water-borne sampling approach. Our results show that A. femoralis males exhibited a positive behavioural and androgenic response after being confronted to simulated territorial intrusions, providing support for the Challenge Hypothesis in a territorial frog.

Two new glassfrogs (Centrolenidae: Hyalinobatrachium) from Ecuador, with comments on the endangered biodiversity of the Andes.

Background: The Tropical Andes is the world's most biodiverse hotspot. This region contains >1,000 amphibian species, more than half of which are endemic. Herein we describe two new glassfrog species (Centrolenidae: Hyalinobatrachium) that we discovered within relatively unexplored and isolated localities of the Ecuadorian Andes. Methods: We employed morphological, acoustic, and molecular methods to test the hypothesis that Hyalinobatrachium mashpi sp. nov and H. nouns sp. nov. are species new to science. Following standard methods, we generated mitochondrial sequences (16S) of 37 individuals in the genus Hyalinobatrachium. We inferred the phylogenetic relationships of the two new species in comparison to all other glassfrogs using Maximum Likelihood. In addition to describing the call of H. mashpi sp. nov., we performed a discriminant analysis of principal components (DAPC) with the advertisement call characteristics of several congeners. Results: Based on an integrative taxonomy approach, we describe two new species. Morphological traits and the inferred phylogeny unambiguously place the new taxa in the genus Hyalinobatrachium. Both species are distinguished from other glassfrogs mainly by their dorsal coloration (i.e., dorsum lime green with small light yellow spots, head usually with interorbital bar) and transparent pericardium (i.e., the heart is visible through the ventral skin). The new species exhibit a high morphological similarity (i.e., cryptic) and occur within relatively close geographical proximity (closest aerial distance = 18.9 km); however, their uncorrected p distance for the mitochondrial gene 16S is 4.6-4.7%, a value that greatly exceeds the genetic distance between closely related species of centrolenid frogs. The DAPC revealed that the advertisement call of H. mashpi sp. nov. is acoustically distinct. Discussion: Our findings are congruent with several previous studies that report a high degree of endemism in the Toisán mountain range, which appears to be isolated from the main Andean cordillera for some amphibian groups. We recommend that both H. mashpi sp. nov. and H. nouns sp. nov. be listed as Endangered, following IUCN criteria. These new species provide another example of cryptic diversity in the Andes-further evidence that the region fosters much more biodiversity than we have the resources to catalog. Threatened by mining and other exploitative industries, these glassfrogs and many other yet-to-be-discovered Andean species highlight the dire need for effective conservation measures-especially in northwestern Ecuador.

Mate choice in a promiscuous poison frog.

In many animal species, members of one sex, most often females, exhibit a strong preference for mating partners with particular traits or resources. However, when females sequentially mate with multiple partners, strategies underlying female choice are not very well understood. Particularly, little is known if under such sequential polyandry females mate truly randomly, or if they actively try to spread mating events across multiple partners. In the present study, we used the highly promiscuous poison frog Allobates femoralis to investigate whether promiscuity could result from a preference for novel mates. Furthermore, we examined the importance of call characteristics for mate choice. We conducted mate choice experiments in a laboratory setup, by presenting females with recent mating partners or novel males. We recorded call characteristics of both males and the time females spent close to each male. In our trials, females preferred previous mating partners over novel males and also males with shorter advertisement calls. Results from previous studies on A. femoralis suggest that females in our trials recognized previous partners based on individual call characteristics. While mating decisions in the wild and in the laboratory might differ, our study provides first evidence for female mate choice in a poison frog with sequential polyandry.

Naive poison frog tadpoles use bi-modal cues to avoid insect predators but not heterospecific predatory tadpoles.

For animals to survive until reproduction, it is crucial that juveniles successfully detect potential predators and respond with appropriate behavior. The recognition of cues originating from predators can be innate or learned. Cues of various modalities might be used alone or in multi-modal combinations to detect and distinguish predators but studies investigating multi-modal integration in predator avoidance are scarce. Here, we used wild, naive tadpoles of the Neotropical poison frog Allobates femoralis ( Boulenger, 1884) to test their reaction to cues with two modalities from two different sympatrically occurring potential predators: heterospecific predatory Dendrobates tinctorius tadpoles and dragonfly larvae. We presented A. femoralis tadpoles with olfactory or visual cues, or a combination of the two, and compared their reaction to a water control in a between-individual design. In our trials, A. femoralis tadpoles reacted to multi-modal stimuli (a combination of visual and chemical information) originating from dragonfly larvae with avoidance but showed no reaction to uni-modal cues or cues from heterospecific tadpoles. In addition, visual cues from conspecifics increased swimming activity while cues from predators had no effect on tadpole activity. Our results show that A. femoralis tadpoles can innately recognize some predators and probably need both visual and chemical information to effectively avoid them. This is the first study looking at anti-predator behavior in poison frog tadpoles. We discuss how parental care might influence the expression of predator avoidance responses in tadpoles.

Experience shapes accuracy in territorial decision-making in a poison frog.

The trade-off between speed and accuracy affects many behavioural processes like predator avoidance, foraging and nest-site selection, but little is known about this trade-off relative to territorial behaviour. Some poison frogs are highly territorial and fiercely repel calling male intruders. However, attacks need to be conducted cautiously, as they are energetically costly and bear the risk of own injury or accidentally targeting the wrong individual. In this study, we investigated the speed-accuracy trade-off in the context of male territoriality during the breeding season in the brilliant-thighed poison frog, Allobates femoralis. In our experiment, we presented the call of an invisible 'threatening' intruder together with a visible 'non-threatening' intruder, using acoustic playback and a frog model, respectively. Contrary to our prediction, neither reaction time nor approach speed of the tested frogs determined the likelihood of erroneous attacks. However, younger individuals were more likely to attack the non-threatening model than older ones, suggesting that experience plays an essential role in identifying and distinguishing rivalling individuals in a territorial context.

Reproductive behavior drives female space use in a sedentary Neotropical frog.

Longer-range movements of anuran amphibians such as mass migrations and habitat invasion have received a lot of attention, but fine-scale spatial behavior remains largely understudied. This gap is especially striking for species that show long-term site fidelity and display their whole behavioral repertoire in a small area. Studying fine-scale movement with conventional capture-mark-recapture techniques is difficult in inconspicuous amphibians: individuals are hard to find, repeated captures might affect their behavior and the number of data points is too low to allow a detailed interpretation of individual space use and time budgeting. In this study, we overcame these limitations by equipping females of the Brilliant-Thighed Poison Frog (Allobates femoralis) with a tag allowing frequent monitoring of their location and behavior. Neotropical poison frogs are well known for their complex behavior and diverse reproductive and parental care strategies. Although the ecology and behavior of the polygamous leaf-litter frog Allobates femoralis is well studied, little is known about the fine-scale space use of the non-territorial females who do not engage in acoustic and visual displays. We tracked 17 females for 6 to 17 days using a harmonic direction finder to provide the first precise analysis of female space use in this species. Females moved on average 1 m per hour and the fastest movement, over 20 m per hour, was related to a subsequent mating event. Traveled distances and activity patterns on days of courtship and mating differed considerably from days without reproduction. Frogs moved more on days with lower temperature and more precipitation, but mating seemed to be the main trigger for female movement. We observed 21 courtships of 12 tagged females. For seven females, we observed two consecutive mating events. Estimated home ranges after 14 days varied considerably between individuals and courtship and mating associated space use made up for ∼30% of the home range. Allobates femoralis females spent large parts of their time in one to three small centers of use. Females did not adjust their time or space use to the density of males in their surroundings and did not show wide-ranging exploratory behavior. Our study demonstrates how tracking combined with detailed behavioral observations can reveal the patterns and drivers of fine-scale spatial behavior in sedentary species.

Cannibalism.

Chloe Fouilloux and colleagues introduce animal cannibalism.

Transcriptomic Signatures of Experimental Alkaloid Consumption in a Poison Frog.

In the anuran family Dendrobatidae, aposematic species obtain their toxic or unpalatable alkaloids from dietary sources, a process known as sequestering. To understand how toxicity evolved in this family, it is paramount to elucidate the pathways of alkaloid processing (absorption, metabolism, and sequestering). Here, we used an exploratory skin gene expression experiment in which captive-bred dendrobatids were fed alkaloids. Most of these experiments were performed with Dendrobates tinctorius, but some trials were performed with D. auratus, D. leucomelas and Allobates femoralis to explore whether other dendrobatids would show similar patterns of gene expression. We found a consistent pattern of up-regulation of genes related to muscle and mitochondrial processes, probably due to the lack of mutations related to alkaloid resistance in these species. Considering conserved pathways of drug metabolism in vertebrates, we hypothesize alkaloid degradation is a physiological mechanism of resistance, which was evidenced by a strong upregulation of the immune system in D. tinctorius, and of complement C2 across the four species sampled. Probably related to this strong immune response, we found several skin keratins downregulated, which might be linked to a reduction of the cornified layer of the epidermis. Although not conclusive, our results offer candidate genes and testable hypotheses to elucidate alkaloid processing in poison frogs.

MEMRI for visualizing brain activity after auditory stimulation in frogs.

Anuran amphibians are common model organisms in bioacoustics and neurobiology. To date, however, most available methods for studying auditory processing in frogs are highly invasive and thus do not allow for longitudinal study designs, nor do they provide a global view of the brain, which substantially limits the questions that can be addressed. The goal of this study was to identify areas in the frog brain that are responsible for auditory processing using in vivo manganese-enhanced MRI (MEMRI). We were interested in determining if the neural processing of socially relevant acoustic stimuli (e.g., species-specific calls) engages a specific pattern of brain activation that differs from patterns elicited by less- or nonrelevant acoustic signals. We thus designed an experiment, in which we presented three different types of acoustic stimuli (species-specific calls, band-limited noise, or silence) to fully awake northern leopard frogs (Rana pipiens) and then conducted MEMRI T1-weighted imaging to investigate differences in signal intensity due to manganese uptake as an indication of brain activity across all three conditions. We found the greatest change in signal intensity within the torus semicircularis (the principal central auditory region), the habenula, and the paraphysis of frogs that had been exposed to conspecific calls compared with noise or silence conditions. Stimulation with noise did not result in the same activation patterns, indicating that signals with contrasting social relevance are differentially processed in these areas of the amphibian brain. MEMRI provides a powerful approach to studying brain activity with high spatial resolution in frogs. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Within-generation and transgenerational plasticity of mate choice in oceanic stickleback under climate change.

Plasticity, both within and across generations, can shape sexual traits involved in mate choice and reproductive success, and thus direct measures of fitness. Especially, transgenerational plasticity (TGP), where parental environment influences offspring plasticity in future environments, could compensate for otherwise negative effects of environmental change on offspring sexual traits. We conducted a mate choice experiment using stickleback ( Gasterosteus aculeatus) with different thermal histories (ambient 17°C or elevated 21°C) within and across generations under simulated ocean warming using outdoor mesocosms. Parentage analysis of egg clutches revealed that maternal developmental temperature and reproductive (mesocosm) environment affected egg size, with females that developed at 17°C laying smaller eggs in 21°C mesocosms, likely owing to metabolic costs at elevated temperature. Paternal developmental temperature interacted with the reproductive environment to influence mating success, particularly under simulated ocean warming, with males that developed at 21°C showing lower overall mating success compared with 17°C males, but higher mating success in 21°C mesocosms. Furthermore, mating success of males was influenced by the interaction between F1 developmental temperature and F0 parent acclimation temperature, demonstrating the potential role of both TGP and within-generation plasticity in shaping traits involved in sexual selection and mate choice, potentially facilitating rapid responses to environmental change. This article is part of the theme issue 'The role of plasticity in phenotypic adaptation to rapid environmental change'.