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Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite life-long neoteny in nature and in captive-bred colonies, metamorphosis of these animals can be experimentally induced by administering Thyroid hormones (THs). However, microbiological consequences of this experimental procedure, such as host microbiota response, remain largely unknown. Here, we systematically compared host bacterial microbiota associated with skin, stomach, gut tissues and fecal samples, between neotenic and metamorphic axolotls based on 16S rRNA gene sequences. Our results show that distinct bacterial communities inhabit individual organs of axolotl and undergo substantial restructuring through metamorphosis. Skin microbiota among others, shifted sharply, as highlighted by a major transition from Firmicutes-enriched to Proteobacteria-enriched relative abundance and precipitously decreased diversity. Fecal microbiota of neotenic and metamorphic axolotl shared relatively higher similarity, suggesting that diet continues to shape microbiota despite fundamental transformations in the host digestive organs. We also reproduced the previous finding on reduction in regenerative capacity in limbs of axolotl following metamorphosis, highlighting the need to investigate whether shifts in microbiota is causally linked to regenerative capacity of axolotl. The initial results on axolotl microbiota provide novel insights into microbiological aspects of axolotl metamorphosis and will establish a baseline for future in-depth studies.

38Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a 39 model organism for regenerative and developmental biology. Despite life-long neoteny in 40 nature and in captive-bred colonies, metamorphosis of these animals can be 41 experimentally induced by administering Thyroid hormones (THs). However, biological 42 consequences of this experimental procedure, such as host microbiota response and 43 implications for regenerative capacity, remain largely unknown. Here, we systematically 44 compared host bacterial microbiota associated with skin, stomach, gut tissues and fecal 45 samples based on 16S rRNA gene sequences, along with limb regenerative capacity, 46 between neotenic and metamorphic Axolotls. Our results show that distinct bacterial 47 communities inhabit individual organs of Axolotl and undergo substantial restructuring 48 through metamorphosis. Drastic restructuring was observed for skin microbiota, 49 highlighted by a major transition from Firmicutes-enriched to Proteobacteria-enriched 50 relative abundance and precipitously decreased diversity. Remarkably, shifts in 51 microbiota was accompanied by a steep reduction in limb regenerative capacity. Fecal 52 microbiota of neotenic and metamorphic Axolotl shared relatively higher similarity, 53suggesting that diet continues to shape microbiota despite fundamental transformations in 54 the host digestive organs. The results provide novel insights into microbiological and 55 regenerative aspects of Axolotl metamorphosis and will establish a baseline for future in-56 depth studies. 57 58 59 60 61 62 63 Metazoan genomes have diversified and evolved in the presence of associated host 64 microbiota. The evolution of morphology and function of animal organ systems may have 65 been influenced by interactions with their microbial partners 1 . From the host perspective, 66 symbioses between metazoans and microbes provide a synergetic impact to operate 67 essential functions for normal growth, development and behavior 2-4 . Studies on host-68 microbiome interactions in health and disease conditions indicate that perturbation of the 69 crosstalk between the host and microorganisms may lead to deleterious consequences 70 such as developmental defects 3,5 , increased susceptibility to infectious diseases 6,7 and 71 ultimately fitness costs 8 . Even though host genotypes 9 and environmental factors, such 72 as diet and habitat 10,11 , were shown to strongly impact the composition and structure of 73 animal microbiota, ecological forces shaping assembly of the host associated microbial 74 communities have still been poorly understood. 75 76 Amphibians, which undergo dramatic morphological changes through metamorphosis, 77 exhibit explicitly altered biphasic life stages to tackle developmental challenges. 78 Remarkably, metamorphosis in several marine animal species such as sponges, corals, 79 crabs, sea urchins, an ascidians is mediated by bacterial community 12 . Thyroid hormones 80 (THs) are key players in initiation and completion of metamor...

SUMMARY:Regeneration is defined as tissue renewal and functional restoration process of the damaged parts of the body after an injury. Ambystoma mexicanum, commonly named the Axolotl, is one of the unique vertebrates, which has a remarkable ability to regenerate their extremities following the amputation. Although the process of regeneration includes several periods, it can be divided into two main phases; blastema formation and dedifferentiation. In the couple of hours following the amputation, wound closure occurs by migration of epithelial cells around the amputation site followed by macrophage infiltration and dedifferentiation of cells to turn into stem cells. Accumulated stem cells form a very authentic tissue type called blastema, which is crucial for successful regeneration. In order to evaluate this exceptional tissue and acquire high quality images, it is crucial to employ specific procedures to prepare the tissue for imaging. Here, in this study, we aimed to investigate success of various fixative solutions (Carnoy's, Bouin's, % 10 NBF, Clarke's, Alcoholic Formaline and AFA) to monitor the fixed blastema. Our data reveals that integrity of the blastema tissue differs among used fixatives and a significant difference is observed between the samples in terms of staining quality.