Induced parental care in a poison frog: a tadpole cross-fostering experiment.

Understanding the external stimuli and natural contexts that elicit complex behaviours, such as parental care, is key in linking behavioural mechanisms to their real-life function. Poison frogs provide obligate parental care by shuttling their tadpoles from terrestrial clutches to aquatic nurseries, but little is known about the proximate mechanisms that control these behaviours. In this study, we used Allobates femoralis, a poison frog with predominantly male parental care, to investigate whether tadpole transport can be induced in both sexes by transferring unrelated tadpoles to the backs of adults in the field. Specifically, we asked whether the presence of tadpoles on an adult's back can override the decision-making rules preceding tadpole pick-up and induce the recall of spatial memory necessary for finding tadpole deposition sites. We used telemetry to facilitate accurate tracking of individual frogs and spatial analysis to compare movement trajectories. All tested individuals transported their foster-tadpoles to water pools outside their home area. Contrary to our expectation, we found no sex difference in the likelihood to transport or in the spatial accuracy of finding tadpole deposition sites. We reveal that a stereotypical cascade of parental behaviours that naturally involves sex-specific offspring recognition strategies and the use of spatial memory can be manipulated by experimental placement of unrelated tadpoles on adult frogs. As individuals remained inside their home area when only the jelly from tadpole-containing clutches was brushed on the back, we speculate that tactile rather than chemical stimuli trigger these parental behaviours.

Disease-specific molecular events in cortical multiple sclerosis lesions.

Cortical lesions constitute an important part of multiple sclerosis pathology. Although inflammation appears to play a role in their formation, the mechanisms leading to demyelination and neurodegeneration are poorly understood. We aimed to identify some of these mechanisms by combining gene expression studies with neuropathological analysis. In our study, we showed that the combination of inflammation, plaque-like primary demyelination and neurodegeneration in the cortex is specific for multiple sclerosis and is not seen in other chronic inflammatory diseases mediated by CD8-positive T cells (Rasmussen's encephalitis), B cells (B cell lymphoma) or complex chronic inflammation (tuberculous meningitis, luetic meningitis or chronic purulent meningitis). In addition, we performed genome-wide microarray analysis comparing micro-dissected active cortical multiple sclerosis lesions with those of tuberculous meningitis (inflammatory control), Alzheimer's disease (neurodegenerative control) and with cortices of age-matched controls. More than 80% of the identified multiple sclerosis-specific genes were related to T cell-mediated inflammation, microglia activation, oxidative injury, DNA damage and repair, remyelination and regenerative processes. Finally, we confirmed by immunohistochemistry that oxidative damage in cortical multiple sclerosis lesions is associated with oligodendrocyte and neuronal injury, the latter also affecting axons and dendrites. Our study provides new insights into the complex mechanisms of neurodegeneration and regeneration in the cortex of patients with multiple sclerosis.

Effects of parental care on skin microbial community composition in poison frogs.

Parent-offspring interactions constitute the first contact of many newborns with their environment, priming community assembly of microbes through priority effects. Early exposure to microbes can have lasting influences on the assembly and functionality of the host's microbiota, leaving a life-long imprint on host health and disease. Studies of the role played by parental care in microbial acquisition have primarily focused on humans and hosts with agricultural relevance. Anuran vertebrates offer the opportunity to examine microbial community composition across life stages as a function of parental investment. In this study, we investigate vertical transmission of microbiota during parental care in a poison frog (Family Dendrobatidae), where fathers transport their offspring piggyback-style from terrestrial clutches to aquatic nurseries. We found that substantial bacterial colonization of the embryo begins after hatching from the vitelline envelope, emphasizing its potential role as microbial barrier during early development. Using a laboratory cross-foster experiment, we demonstrated that poison frogs performing tadpole transport serve as a source of skin microbes for tadpoles on their back. To study how transport impacts the microbial skin communities of tadpoles in an ecologically relevant setting, we sampled frogs and tadpoles of sympatric species that do or do not exhibit tadpole transport in their natural habitat. We found more diverse microbial communities associated with tadpoles of transporting species compared to a non-transporting frog. However, we detected no difference in the degree of similarity between adult and tadpole skin microbiotas, based on whether the frog species exhibits transporting behavior or not. Using a field experiment, we confirmed that tadpole transport can result in the persistent colonization of tadpoles by isolated microbial taxa associated with the caregiver's skin, albeit often at low prevalence. This is the first study to describe vertical transmission of skin microbes in anuran amphibians, showing that offspring transport may serve as a mechanism for transmission of parental skin microbes. Overall, these findings provide a foundation for further research on how vertical transmission in this order impacts host-associated microbiota and physiology.

Home security cameras as a tool for behavior observations and science equity.

Reliably capturing transient animal behavior in the field and laboratory remains a logistical and financial challenge, especially for small ectotherms. Here, we present a camera system that is affordable, accessible, and suitable to monitor small, cold-blooded animals historically overlooked by commercial camera traps, such as small amphibians. The system is weather-resistant, can operate offline or online, and allows collection of time-sensitive behavioral data in laboratory and field conditions with continuous data storage for up to four weeks. The lightweight camera can also utilize phone notifications over Wi-Fi so that observers can be alerted when animals enter a space of interest, enabling sample collection at proper time periods. We present our findings, both technological and scientific, in an effort to elevate tools that enable researchers to maximize use of their research budgets. We discuss the relative affordability of our system for researchers in South America, which is home to the largest population of ectotherm diversity.

Binding and sequestration of poison frog alkaloids by a plasma globulin.

Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid-binding molecule in blood of poison frogs. Proteomic and biochemical studies establish this plasma protein to be a liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid-binding activity, ABG sequesters and regulates the bioavailability of 'free' plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin-binding proteins. ABG represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points toward serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.

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.

Pathogenic T cell responses against aquaporin 4.

Inflammatory lesions in the central nervous system of patients with neuromyelitis optica are characterized by infiltration of T cells and deposition of aquaporin-4-specific antibodies and complement on astrocytes at the glia limitans. Although the contribution of aquaporin-4-specific autoantibodies to the disease process has been recently elucidated, a potential role of aquaporin-4-specific T cells in lesion formation is unresolved. To address this issue, we raised aquaporin-4-specific T cell lines in Lewis rats and characterized their pathogenic potential in the presence and absence of aquaporin-4-specific autoantibodies of neuromyelitis optica patients. We show that aquaporin-4-specific T cells induce brain inflammation with particular targeting of the astrocytic glia limitans and permit the entry of pathogenic anti-aquaporin-4-specific antibodies to induce NMO-like lesions in spinal cord and brain. In addition, transfer of aquaporin-4-specific T cells provoked mild (subclinical) myositis and interstitial nephritis. We further show that the expression of the conformational epitope, recognized by NMO patient-derived aquaporin-4-specific antibodies is induced in kidney cells by the pro-inflammatory cytokine gamma-interferon. Our data provide further support for the view that NMO lesions may be induced by a complex interplay of T cell mediated and humoral immune responses against aquaporin-4.

Pool choice in a vertical landscape: Tadpole-rearing site flexibility in phytotelm-breeding frogs.

Many species of Neotropical frogs have evolved to deposit their tadpoles in small water bodies inside plant structures called phytotelmata. These pools are small enough to exclude large predators but have limited nutrients and high desiccation risk. Here, we explore phytotelm use by three common Neotropical species: Osteocephalus oophagus, an arboreal frog that periodically feeds eggs to its tadpoles; Dendrobates tinctorius, a tadpole-transporting poison frog with cannibalistic tadpoles; and Allobates femoralis, a terrestrial tadpole-transporting poison frog with omnivorous tadpoles. We found that D. tinctorius occupies pools across the chemical and vertical gradient, whereas A. femoralis and O. oophagus appear to have narrower deposition options that are restricted primarily by pool height, water capacity, alkalinity, and salinity. Dendrobates tinctorius tadpoles are particularly flexible and can survive in a wide range of chemical, physical, and biological conditions, whereas O. oophagus seems to prefer small, clear pools and A. femoralis occupies medium-sized pools with abundant leaf litter and low salinity. Together, these results show the possible niche partitioning of phytotelmata among frogs and provide insight into stressors and resilience of phytotelm breeders.

Inflammation induced by innate immunity in the central nervous system leads to primary astrocyte dysfunction followed by demyelination.

Primary loss and dysfunction of astrocytes may trigger demyelination, as seen in neuromyelitis optica, an inflammatory disease of the central nervous system. In most patients affected by this disease, injury to astrocytes is initiated by the action of autoantibodies targeting aquaporin 4 (AQP-4), a water channel on astrocytes. We show here that damage of astrocytes and subsequent demyelination can also occur in the absence of autoantibody-mediated mechanisms. Following injection of lipopolysaccharide into the white matter initial microglia activation is followed by a functional disturbance of astrocytes, mainly reflected by retraction of astrocytic foot processes at the glia limitans and loss of AQP-4 and connexins, which are involved in the formation of gap junctions between astrocytes and oligodendrocytes. Demyelination and oligodendrocyte degeneration in this model follows astrocyte pathology. Similar structural abnormalities were also seen in a subset of active lesions in multiple sclerosis. Our studies suggest that astrocyte injury may be an important early step in the cascade of lesion formation in brain inflammation.

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.

The "window of susceptibility" for inflammation in the immature central nervous system is characterized by a leaky blood-brain barrier and the local expression of inflammatory chemokines.

Early in postnatal development, the immature central nervous system (CNS) is more susceptible to inflammation than its adult counterpart. We show here that this "window of susceptibility" is characterized by the presence of leaky vessels in the CNS, and by a global chemokine expression profile which is clearly distinct from the one observed in the adult CNS and has three important characteristics. First, it contains chemokines with known roles in the differentiation and maturation of glia and neurons. Secondly, these chemokines have been described before in inflammatory lesions of the CNS, where they are important for the recruitment of monocytes and T cells. Lastly, the chemokine profile is shaped by pathological changes like oligodendrocyte stress and attempts of myelin repair. Changes in the chemokine expression profile along with a leaky blood-brain barrier pave the ground for an accelerated development of CNS inflammation.

After injection into the striatum, in vitro-differentiated microglia- and bone marrow-derived dendritic cells can leave the central nervous system via the blood stream.

The prototypic migratory trail of tissue-resident dendritic cells (DCs) is via lymphatic drainage. Since the central nervous system (CNS) lacks classical lymphatic vessels, and antigens and cells injected into both the CNS and cerebrospinal fluid have been found in deep cervical lymph nodes, it was thought that CNS-derived DCs exclusively used the cerebrospinal fluid pathway to exit from tissues. It has become evident, however, that DCs found in peripheral organs can also leave tissues via the blood stream. To study whether DCs derived from microglia and bone marrow can also use this route of emigration from the CNS, we performed a series of experiments in which we injected genetically labeled DCs into the striata of rats. We show here that these cells migrated from the injection site to the perivascular space, integrated into the endothelial lining of the CNS vasculature, and were then present in the lumen of CNS blood vessels days after the injection. Moreover, we also found these cells in both mesenteric lymph nodes and spleens. Hence, microglia- and bone marrow-derived DCs can leave the CNS via the blood stream.

T cell-activation in neuromyelitis optica lesions plays a role in their formation.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system (CNS), which is characterized by the presence of pathogenic serum autoantibodies against aquaporin 4 (AQP4) in the vast majority of patients. The contribution of T cells to the formation of astrocyte destructive lesions is currently unclear. However, active human NMO lesions contain CD4+ T-lymphocytes expressing the activation marker Ox40, and the expression is more profound compared to that seen in MS lesions of comparable activity. Therefore, we analyzed the role of T-cell activation within the CNS in the initiation of NMO lesions in an experimental model of co-transfer of different encephalitogenic T-cells and human AQP4 antibody containing NMO immunoglobulin (NMO IgG). We further studied the expression of the T-cell activation marker Ox40 in NMO and multiple sclerosis lesions in different stages of activity. RESULTS: All encephalitogenic T-cell lines used in our experiments induced brain inflammation with a comparable extent of blood brain barrier damage, allowing human NMO IgG to penetrate into the brain and spinal cord tissue. However, astrocyte destructive NMO lesions were only seen with T-cells, which showed signs of activation in the lesions. T-cell activation was reflected by the expression of the activation marker Ox40 and pronounced production of γ-IFN, which was able to increase the production of complement proteins and of the Fc gamma III receptor (Fcgr3) and decreased production of complement inhibitory protein Factor H in microglia. CONCLUSIONS: Our data indicate that local activation of T-cells provide an inflammatory environment in the CNS, which allows AQP4 auto-antibodies to induce astrocyte destructive NMO-like lesions.

Prospects of transcript profiling for mRNAs and MicroRNAs using formalin-fixed and paraffin-embedded dissected autoptic multiple sclerosis lesions.

The elaboration of novel pathogenic aspects of multiple sclerosis (MS) requires the analysis of well-defined stages of lesion development. However, specimens of certain stages and lesion types are either present in small brain biopsies, insufficient in size for further molecular studies or available as formalin-fixed and paraffin-embedded (FFPE) material only. Therefore, application of current molecular biology techniques to FFPE tissue is warranted. We compared FFPE and frozen tissue by using quantitative polymerase chain reaction and report: (1) FFPE material is highly heterogeneous regarding the utility for transcript profiling of mRNAs; well-preserved FFPE samples had about a 100-fold reduced sensitivity compared with frozen tissue, but gave similar results for genes of sufficient abundance; (2) FFPE samples not suitable for mRNA analysis are still highly valuable for miRNA quantification; (3) the length of tissue fixation greatly affects utility for mRNA but not for miRNA analysis; (4) FFPE samples can be processed via a hot water bath for dissection of defined lesion areas; and (5) in situ hybridization for proteolipid protein (PLP) helps to identify samples not suitable for mRNA amplification. In summary, we present a detailed protocol how to use autoptic FFPE tissue for transcript profiling in dissected tissue areas.