Expression of Glycogenic Genes in the Oviduct of Chinese Brown Frog (Rana Dybowskii) During Pre-Hibernation

Background: The oviduct of Chinese brown frog (Rana dybowskii) expands specifically during pre-hibernation. However, the underlying molecular mechanism remains unclear. Results: This study investigated the mRNA and protein expression levels of glycogenic genes in the oviduct of Rana dybowskii during the breeding season and pre-hibernation. Morphological studies revealed increased weight and enlarged diameter of the oviduct in pre-hibernation. Furthermore, significantly increased glycogen level was detected in the oviduct during pre-hibernation when compared to the breeding season. Transcriptome analysis further identified that the differentially expressed genes in the synthesis and metabolism pathways of carbohydrates in the oviduct during pre-hibernation. The relative mRNA levels of glycolysis and glycogenesis-related genes were up-regulated, while gluconeogenesis-related genes were down-regulated during pre-hibernation. Also, Western blot showed that glucose transporter and glycogen synthesis-related proteins, such as total-GYS, and p-GSK-3β were highly expressed in the oviduct during pre-hibernation. In addition, immunohistochemical data showed that glucose transporter and total-GYS, p-GYS, total-GSK-3β and p-GSK-3β related to glycogen synthesis were both expressed regionally in the oviduct of Rana dybowskii during the breeding season and pre-hibernation. Conclusions: The data suggests that carbohydrate metabolism plays an important role in the hibernation process of Rana dybowskii, and provides a new insight into the mechanism underlying the expansion of the oviduct of Rana dybowskii during pre-hibernation.

Immunoreactivities of AR, ERα, ERβ and aromatase in the nuptial pad of Chinese brown frog (Rana dybowskii) during pre-hibernation and the breeding period

There is a prominent local raised pad called nuptial pad on the forelimb of Chinese brown frog (Rana dybowskii), which is hypothetically concluded as an enhancement of the grip and a spreader of pheromone during the amplexus. In this study, we investigated the immunolocalization and protein expression levels of AR, ERα, ERβ and aromatase in the nuptial pad of R. dybowskii during pre-hibernation and the breeding period. Histologically, the annual development of the nuptial pad in R. dybowskii is manifested as the larger area of specialized mucous gland and the longer length of papillary epidermal projection during the breeding period. AR, ERα, ERβ and aromatase are present in the stratum granulosum, stratum spinosum, stratum basale and the secretory portion of specialized mucous glands during both periods. Western blotting results confirmed that AR, ERα and ERβ protein levels are higher during pre-hibernation than those during the breeding season. These results suggest that nuptial pad is the direct target organ of androgen and estrogen. Androgen may participate in the regulation of annual development and glandular function of nuptial pad, and estrogen may play an endocrine, autocrine or paracrine role during pre-hibernation and the breeding period.

Changes in the Gut Microbiota Diversity of Brown Frogs (Rana Dybowskii) After Local Antimicrobial Therapy

Background: Amphibians frequently receive an antibiotic bath after feedlot placement to control bacterial diseases. The potential collateral effect of these antibiotics on the frog microbiota is largely unknown. Antibiotics are frequently employed to examine the role of the gut microbiota. Existing research relies mainly on oral antibiotics, but knowledge regarding the effects of antibiotics on the gut microbiota through a bath or local antimicrobial therapies is limited. Results: The gut microbiota of gentamicin, recovered, and control Rana dybowskii groups were compared by Illumina high-throughput sequencing, and the functional profiles were analysed using a phylogenetic investigation of communities by the reconstruction of unobserved states (PICRUSt). Furthermore, the relationship between gut microbiota structures and forecast function compositions was determined. The results showed that the alpha diversity indices were significantly reduced by the gentamicin bath, which significantly changed the composition of the gut microbiota. After 7 days, the gut microbiota was still similar to that during the gentamicin bath. Forty-four indicator species were selected at the genus level, namely, 42 species indicating the control group and 2 species indicating the gentamicin and recovery groups. Potential pathogenic bacteria belonging to Aeromonas, Citrobacter, and Chryseobacterium significantly decreased after the gentamicin bath. The community similarity assays did not show an obvious discrepancy in the functional composition between the gentamicin and control frogs, indicating that the functions of the gut bacterial community were highly redundant. Conclusions: The gentamicin bath significantly reduced the alpha diversity of the gut microbiota of R. dybowskii. Gentamicin significantly changed the structure of the gut microbiota, and the gut microbiotas exhibited weak resilience and did not totally recover after seven days. The gentamicin bath did not change the functional composition of the gut microbiota of R. dybowskii, and there was no significant correlation between the composition of the gut microbiota and the functional composition, illustrating the high intestinal functional redundancy of the frog gut bacterial community. This work offers basic data for upcoming research, including the establishment of the amphibian gut microbiota and local antibiotic administration, and has important implications for aquaculture management and amphibian conservation.

First detection of ranavirus in a wild population of Dybowski’s brown frog (Rana dybowskii) in South Korea

Background Ranavirus is an emerging infectious disease which has been linked to mass mortality events in various amphibian species. In this study, we document the first mass mortality event of an adult population of Dybowski’s brown frogs (Rana dybowskii), in 2017, within a mountain valley in South Korea. Results We confirmed the presence of ranavirus from all collected frogs (n = 22) via PCR and obtained the 500 bp major capsid protein (MCP) sequence from 13 individuals. The identified MCP sequence highly resembled Frog virus 3 (FV3) and was the same haplotype of a previously identified viral sequence collected from Huanren brown frog (R. huanrenensis) tadpoles in South Korea. Human habitat alteration, by recent erosion control works, may be partially responsible for this mass mortality event. Conclusion We document the first mass mortality event in a wild Korean population of R. dybowskii. We also suggest, to determine if ranavirus infection is a threat to amphibians, government officials and researchers should develop continuous, country-wide, ranavirus monitoring programs of Korean amphibian populations.