Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles vis lipopolysaccharide signalling

Xenopus laevis tadpoles can regenerate functional tails, containing spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around one week of age. At this stage, amputation of the tadpole's tail may either result in scarless wound healing, or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles, and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Knocking down TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect.

Early life skin microbial trajectory as a function of vertical and environmental transmission in Bornean foam-nesting frogs

Background The amphibian skin microbiome is an important mediator of host health and serves as a potential source of undiscovered scientifically significant compounds. However, the underlying modalities of how amphibian hosts obtain their initial skin-associated microbiome remains unclear. Here, we explore microbial transmission patterns in foam-nest breeding tree frogs from Southeast Asia (Genus: Polypedates) whose specialized breeding strategy allows for better delineation between vertically and environmentally derived microbes. To facilitate this, we analyzed samples associated with adult frog pairs taken after mating—including adults of each sex, their foam nests, environments, and tadpoles before and after environmental interaction—for the bacterial communities using DNA metabarcoding data (16S rRNA). Samples were collected from frogs in-situ in Brunei, Borneo, a previously unsampled region for amphibian-related microbial diversity. Results Adult frogs differed in skin bacterial communities among species, but tadpoles did not differ among species. Foam nests had varying bacterial community composition, most notably in the nests’ moist interior. Nest interior bacterial communities were discrete for each nest and overall displayed a narrower diversity compared to the nest exteriors. Tadpoles sampled directly from the foam nest displayed a bacterial composition less like the nest interior and more similar to that of the adults and nest exterior. After one week of pond water interaction the tadpole skin microbiome shifted towards the tadpole skin and pond water microbial communities being more tightly coupled than between tadpoles and the internal nest environment, but not to the extent that the skin microbiome mirrored the pond bacterial community. Conclusions Both vertical influence and environmental interaction play a role in shaping the tadpole cutaneous microbiome. Interestingly, the interior of the foam nest had a distinct bacterial community from the tadpoles suggesting a limited environmental effect on tadpole cutaneous bacterial selection at initial stages of life. The shift in the tadpole microbiome after environmental interaction indicates an interplay between underlying host and ecological mechanisms that drive community formation. This survey serves as a baseline for further research into the ecology of microbial transmission in aquatic animals.

A ‘tad’ of hope in the fight against airway disease

Xenopus tadpoles have emerged as a powerful in vivo model system to study mucociliary epithelia such as those found in the human airways. The tadpole skin has mucin-secreting cells, motile multi-ciliated cells, ionocytes (control local ionic homeostasis) and basal stem cells. This cellular architecture is very similar to the large airways of the human lungs and represents an easily accessible and experimentally tractable model system to explore the molecular details of mucociliary epithelia. Each of the cell types in the tadpole skin has a human equivalent and a conserved network of genes and signalling pathways for their differentiation has been discovered. Great insight into the function of each of the cell types has been achieved using the Xenopus model and this has enhanced our understanding of airway disease. This simple model has already had a profound impact on the field but, as molecular technologies (e.g. gene editing and live imaging) continue to develop apace, its use for understanding individual cell types and their interactions will likely increase. For example, its small size and genetic tractability make it an ideal model for live imaging of a mucociliary surface especially during environmental challenges such as infection. Further potential exists for the mimicking of human genetic mutations that directly cause airway disease and for the pre-screening of drugs against novel therapeutic targets.

Transient integumentary structures in Boana riojana (Anura, Hylidae) tadpoles

Few studies focusing on embryos and/or tadpole skin morphology have described sensory transient organs whose morphological variation could indicate some taxonomical or functional correlations. We explore here some integumentary features of Boana riojana larvae that are rarely mentioned in tadpole descriptions. We provide histomorphological and SEM descriptions of the lateral line system, a series of evenly distributed unpigmented spots, and some symmetrical paired structures dorsal to the oral disc. The latter are previously unreported in any tadpole. Our descriptions reveal that the: 1) the number of lateral lines resembles those for most tadpoles, but with an unusual arrangement of stitches; 2) paired lateral spots are formed by ciliated cells dispersed in clusters unrelated to the lateral line system; and 3) upper-lip related structures are histomorphologically similar to the unpigmented spots. We discuss and suggest that integumentary transient structures in B. riojana represent traits that should be taken into account when describing tadpoles. This new information may help diagnose species and advance our understanding of tadpole ecomorphology and evolution.

Direct and Indirect Horizontal Transmission of the Antifungal Probiotic Bacterium Janthinobacterium lividum on Green Frog (Lithobates clamitans) Tadpoles

ABSTRACTAmphibian populations worldwide are being threatened by the disease chytridiomycosis, which is caused byBatrachochytrium dendrobatidis. To mitigate the effects ofB. dendrobatidis, bioaugmentation of antifungal bacteria has been shown to be a promising strategy. One way to implement bioaugmentation is through indirect horizontal transmission, defined as the transfer of bacteria from a host to the environment and to another host. In addition, direct horizontal transmission among individuals can facilitate the spread of a probiotic in a population. In this study, we tested whether the antifungal bacteriumJanthinobacterium lividumcould be horizontally transferred, directly or indirectly, in a laboratory experiment usingLithobates clamitanstadpoles. We evaluated the ability ofJ. lividumto colonize the tadpoles' skin and to persist through time using culture-dependent and culture-independent techniques. We also tested whether the addition ofJ. lividumaffected the skin community inL. clamitanstadpoles. We found that transmission occurred rapidly by direct and indirect horizontal transmission, but indirect transmission that included a potential substrate was more effective. Even thoughJ. lividumcolonized the skin, its relative abundance on the tadpole skin decreased over time. The inoculation ofJ. lividumdid not significantly alter the skin bacterial diversity ofL. clamitanstadpoles, which was dominated byPseudomonas. Our results show that indirect horizontal transmission can be an effective bioaugmentation method. Future research is needed to determine the best conditions, including the presence of substrates, under which a probiotic can persist on the skin so that bioaugmentation becomes a successful strategy to mitigate chytridiomycosis.

Cloning and characterization of a functional P2X receptor from larval bullfrog skin

ATP activates an apical-to-basolateral nonselective cation current across the skin of larval bullfrogs ( Rana catesbeiana) with similarities to currents carried by some P2X receptors. A functional P2X receptor was cloned from tadpole skin RNA that encodes a 409-amino acid protein with highest protein homology to cP2X8. RT-PCR showed that this transcript was found in skin, heart, eye, brain, and skeletal muscle of tadpoles but not in skin, brain, or heart of adults. After transcribed RNA from this clone was injected into Xenopus oocytes, application of ATP activated a transient current similar to other P2X receptors and the ATP-activated transient in short-circuit current ( I sc) across intact skin. The agonists 2-methylthio-ATP and adenosine-5′- O-(thiotriphoshate) also activated transient currents. α,β-Methylene-ATP and ADP were poor agonists of this receptor. Suramin and pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS) were potent antagonists, and PPADS showed an irreversible blockade of this receptor to agonist activation. Under external Na+-free, Ca2+/Mg2+-free conditions ( N-methyl-d-glucamine replacement, 0.5 mM EGTA), ATP activated a steadily increasing inward current. Fluorescence microscopy showed that propidium was entering the cells, suggesting that a relatively large pore size was formed under zero divalent conditions. This clone has some characteristics consistent with previously described ATP-activated I sc in the tadpole skin. Because the clone is not found in adult skin, it may have some exclusive role in the tadpole such as sensory reception by the skin or triggering apoptosis at metamorphosis.

Prolactin inhibits corticoid-induced differentiation of active Na+ transport across cultured frog tadpole skin

Active Na+ transport differentiates in larval bullfrog skin cultured with corticoids. After 2 wk in culture, the epidermis became positive against human blood group antigen A, the marker for the adult-type cells of the epidermis, but was negative to the antibody against the acetylcholine receptor, the marker for the larval-type epidermis. Amiloride (10(-5) M) did not inhibit the differentiation of active Na+ transport. On the other hand, in skin cultured with prolactin (2 micrograms/ml), the epidermis remained negative against antigen A and positive against acetylcholine receptor, and the differentiation of active Na+ transport was inhibited. Thyroid hormone did not antagonize the inhibitory action of prolactin on this transport differentiation. Prolactin affected the basal cells of the larval epidermis and inhibited development of corticoid-induced adult features in the epidermis.

Low-affinity mixed acetylcholine-responsive receptors at the apical membrane of frog tadpole skin

The larval frog skin has a very high electrical resistance and a corresponding low rate of transepithelial ion transport. Amiloride, a blocker of sodium transport in adult skin, transiently stimulates rather than inhibits short-circuit current (Isc) across larval skin through nonselective cation channels. Acetylcholine (ACh) stimulates Isc like amiloride, although the response is more prolonged. Pretreatment with ACh markedly suppressed amiloride stimulation of Isc; amiloride pretreatment also suppressed ACh stimulation. Half-maximal stimulation of Isc by ACh occurred at 347 microM. Stimulation by ACh was inhibited by both d-tubocurarine [dissociation constant (Kd) = 57 microM] and atropine (Kd = 49 microM). The specific nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium and the specific muscarinic agonist oxotremorine-M both stimulated Isc and were blocked by either atropine or d-tubocurarine. Reciprocal desensitization and blocker cross-reactivity suggest that ACh activates the same population of receptors as amiloride. This ACh-responsive receptor has characteristics of both nicotinic and muscarinic receptors found in other tissues.