Changes in the gut microbiota diversity of brown frogs (Rana dybowskii) after an antibiotic bath.

BACKGROUND: Captive amphibians frequently receive antibiotic baths to control bacterial diseases. The potential collateral effect of these antibiotics on the microbiota of frogs is largely unknown. To date, studies have mainly relied on oral administration to examine the effects of antibiotics on the gut microbiota; in contrast, little is known regarding the effects of bath-applied antibiotics on the gut microbiota. The gut microbiota compositions of the gentamicin, recovery, and control groups were compared by Illumina high-throughput sequencing, and the functional profiles were analysed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Furthermore, the relationship between the structure and predicted functional composition of the gut microbiota was determined. RESULTS: The alpha diversity indices were significantly reduced by the gentamicin bath, illustrating that this treatment significantly changed the composition of the gut microbiota. After 7 days, the gut microbiota of the recovery group was not significantly different from that of the gentamicin group. Forty-four indicator taxa were selected at the genus level, comprising 42 indicators representing the control group and 2 indicators representing the gentamicin and recovery groups. Potential pathogenic bacteria of the genera Aeromonas, Citrobacter, and Chryseobacterium were significantly depleted after the gentamicin bath. There was no significant positive association between the community composition and functional composition of the gut microbiota in the gentamicin or control frogs, indicating that the functional redundancy of the gut bacterial community was high. CONCLUSIONS: Gentamicin significantly changed the structure of the gut microbiota of R. dybowskii, and the gut microbiota exhibited weak resilience. However, the gentamicin bath did not change the functional composition of the gut microbiota of R. dybowskii, and there was no significant correlation between the structural composition and the functional composition of the gut microbiota.

Effects of Seasonal Hibernation on the Similarities Between the Skin Microbiota and Gut Microbiota of an Amphibian (Rana dybowskii).

Both the gut and skin microbiotas have important functions for amphibians. The gut microbiota plays an important role in both the health and evolution of the host species, whereas the role of skin microbiota in disease resistance is particularly important for amphibians. Many studies have examined the effects of environmental factors on the skin and gut microbiotas, but no study has yet explored the similarities between the skin and gut microbiotas. In this study, the gut and skin microbiotas of Rana dybowskii in summer and winter were investigated via high-throughput Illumina sequencing. The results showed that the alpha diversity of gut and skin microbiotas decreased significantly from summer to winter. In both seasons, the microbial composition and structure differed significantly between the gut and skin, and the similarities between these microbiotas differed between seasons. The pairwise distances between the gut and skin microbiotas were greater in winter than in summer. The ratio of core OTUs and shared OTUs to the sum of the OTUs in the gut and skin microbiotas in summer was significantly higher than that in winter. The similarities between the gut and skin microbiotas are important for understanding amphibian ecology and life history.

Effects of fine-scale habitat quality on activity, dormancy, habitat use, and survival after reproduction in Rana dybowskii (Chordata, Amphibia).

Amphibians are facing population declines and extinctions, and protecting and supplementing refuges can help species survive. However, the microhabitat requirements of most species are unknown, and artificial shelters or burrows have not been well tested for amphibians. Some amphibians exhibit complex behaviour during the transition from post-reproductive dormancy to activity. However, little is known about the ecology, post-reproductive dormancy, and terrestrial activity of amphibians. Here, habitat site selection in experimental enclosures and the effects of shelters (stones, soil) and shade (with and without shade netting) on the activity, exposed body percentage, burrow depth, body-soil contact percentage, and survival of Rana dybowskii were investigated during post-reproductive dormancy and post-dormant activity. The results showed that R. dybowskii live individually under leaves, soil, stones or tree roots. Furthermore, although the dormant sites of frogs are significantly different, the distribution of male and female frogs in these sites is similar. Shading and shelter significantly affected the exposed body percentage, burrow depth and body-soil contact percentage of frogs compared with soil. In the stone group, soil and stone form the frog's refuge/burrow, whereas in the soil group, the refuge/burrow is composed entirely of soil. Even though the soil group has a deeper burrow and a larger area of soil contact with the body, it still has a higher exposure rate than the stone group. Frog activity frequency was affected by shelter and shade; the interaction of shelter and time and the interaction of shading and time were significant. The soil group had a higher activity frequency than the stone group, and the no-shade group had a higher activity frequency than the shade group. Shelter and shading differences do not significantly affect frog survival; however, the death rate during post-reproductive dormancy is lower than that during the active period.

Characteristics and a comparison of the gut microbiota in two frog species at the beginning and end of hibernation.

Season has been suggested to contribute to variation in the gut microbiota of animals. The complicated relationships between amphibians and their gut microbiota and how they change throughout the year require more research. Short-term and long-term hypothermic fasting of amphibians may affect gut microbiota differently; however, these changes have not been explored. In this study, the composition and characteristics of the gut microbiota of Rana amurensis and Rana dybowskii during summer, autumn (short-term fasting) and winter (long-term fasting) were studied by high-throughput Illumina sequencing. Both frog species had higher gut microbiota alpha diversity in summer than autumn and winter, but no significant variations between autumn and spring. The summer, autumn, and spring gut microbiotas of both species differed, as did the autumn and winter microbiomes. In summer, autumn and winter, the dominant phyla in the gut microbiota of both species were Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. All animals have 10 OTUs (>90% of all 52 frogs). Both species had 23 OTUs (>90% of all 28 frogs) in winter, accounting for 47.49 ± 3.84% and 63.17 ± 3.69% of their relative abundance, respectively. PICRUSt2 analysis showed that the predominant functions of the gut microbiota in these two Rana were focused on carbohydrate metabolism, Global and overview maps, Glycan biosynthesis metabolism, membrane transport, and replication and repair, translation. The BugBase analysis estimated that among the seasons in the R. amurensis group, Facultatively_Anaerobic, Forms_Biofilms, Gram_Negative, Gram_Positive, Potentially_Pathogenic were significantly different. However, there was no difference for R. dybowskii. The research will reveal how the gut microbiota of amphibians adapts to environmental changes during hibernation, aid in the conservation of endangered amphibians, particularly those that hibernate, and advance microbiota research by elucidating the role of microbiota under various physiological states and environmental conditions.

Environmental and host factors shaping the gut microbiota diversity of brown frog Rana dybowskii.

Symbiotic microbial communities are common in amphibians, and the composition of gut microbial communities varies with factors such as host phylogeny, life stage, ecology, and diet. However, little is known regarding how amphibians acquire their microbiota or how their growth, development, and environmental factors affect the diversity of their microbiotas. We sampled the gut microbiota during different developmental stages of brown frog Rana dybowskii, including tadpoles (T), frogs in metamorphosis (M), frogs just post-metamorphosis and after eating (F), juvenile frogs in summer (Js), adult frogs in summer (As), adult frogs in autumn (Aa), and hibernating frogs (Ah). We recorded data on the environmental (ambient temperature, fasting status, habitat, and season) and host (body mass and developmental period) factors. We investigated whether the gut microbiota diversity of R. dybowskii differs according to the host developmental stage via high-throughput Illumina sequencing and whether the gut microbiota diversity is affected by environmental and host factors. We found that alpha and beta diversity varied significantly during different developmental stages. The linear discriminant analysis effect size (LEfSe) analysis identified eight phyla exhibiting significant differences: Cyanobacteria (T group), Proteobacteria (M group), Fusobacteria (F group), Firmicutes (As group), Actinobacteria (Aa group), Verrucomicrobia (Aa group), Tenericutes (Aa group), and Bacteroidetes (Ah group). The Venn diagrams showed that 49 shared OTUs were present during all stages of development, whereas 10 OTUs were present in >90% of the samples. The environmental and host factors were significantly correlated with microbial community changes. Furthermore, the AIC-based model results suggested that development was the only variable that needed inclusion in the redundancy analysis (RDA) to explain the variance in taxa. These results have broad implications for our understanding of gut microbiota development and its associations with amphibian development and environmental factors.

Comparison of Gut Microbiota Diversity and Predicted Functions Between Healthy and Diseased Captive Rana dybowskii.

The gut microbiota plays a key role in host health, and disruptions to gut bacterial homeostasis can cause disease. However, the effect of disease on gut microbiota assembly remains unclear and gut microbiota-based predictions of health status is a promising yet poorly established field. Using Illumina high-throughput sequencing technology, we compared the gut microbiota between healthy (HA and HB) and diarrhoeic (DS) Rana dybowskii groups and analyzed the functional profiles through a phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis. In addition, we estimated the correlation between gut microbiota structures and predicted the functional compositions. The results showed significant differences in the phylogenetic diversity (Pd), Shannon, and observed richness (Sobs) indices between the DS and HB groups, with significant differences observed in the gut microbiota composition between the DS group and the HA and HB groups. Linear discriminant analysis (LDA) effect size (LEfSe) results revealed that Proteobacteria were significantly enriched in the DS group; Bacteroidetes were significantly enriched in the HA and HB groups; and Aeromonas, Citrobacter, Enterococcus, Hafnia-Obesumbacterium, Morganella, Lactococcus, Providencia, Vagococcus, and Staphylococcus were significantly enriched in the DS group. Venn diagrams revealed that there were many more unique genera in the DS group than the HA and HB groups. Among 102 sensitive species selected using the indicator method, 33 indicated a healthy status and 69 (e.g., Acinetobacter, Aeromonas, Legionella, Morganella, Proteus, Providencia, Staphylococcus, and Vagococcus) indicated a diseased status. There was a significant and positive association between the composition and functional composition of the gut microbiota, thus indicating low functional redundancy of the frog gut bacterial community. Rana dybowskii disease was associated with changes in the gut microbiota, which subsequently disrupted bacterial-mediated functions. The results of this study can aid in revealing the effect of the R. dybowskii gut microbiota on host health and provide a basis for elucidating the mechanism of the occurrence of R. dybowskii disease.

Effects of Captivity and Season on the Gut Microbiota of the Brown Frog (Rana dybowskii).

The gut microbiota of amphibians is affected by exogenous and endogenous factors. We performed a comprehensive analysis using high-throughput sequencing technology and functional predictions and observed general changes in the gut microbiota of frogs in different growth stages, seasons, and growth environments. There were no significant differences in microbial richness and diversity between juvenile and adult wild frogs, between the summer and autumn groups of captive frogs, or between wild and captive frogs. There were significant differences in the gut microbiota community structure of Rana dybowskii between the summer and autumn groups of captive frogs and between wild and captive R. dybowskii, whereas the differences between juvenile and adult wild frogs were not significant. The dominant gut bacterial phyla in frogs from both captive and wild environments included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Linear discriminant effect size (LEfSe) analysis showed that Bacteroidetes and Firmicutes were significantly enriched in captive and wild R. dybowskii, respectively linear discriminant analysis (LDA > 4). The core operational taxonomical units (OTUs) that were found in >90% of all frogs tested encompassed 15 core OTUs. The captive frogs exhibited 15 core OTUs in addition to the above overall core microbiota, whereas the wild frogs exhibited 19 core OTUs in addition to the above overall core microbiota. Predictions made using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) suggested that eleven KEGG pathways, such as infectious diseases, immune system diseases, metabolism, metabolism of other amino acids, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, neurodegenerative diseases, and transport and catabolism, were enriched in captive frogs. The relative abundance of several red-leg-syndrome-related pathogens increased significantly in captive frogs compared with that in wild frogs. To our knowledge, this is the first study on the effects of individual seasons and captivity on the gut microbiota of frogs.

Comparison of the gut microbiota of Rana amurensis and Rana dybowskii under natural winter fasting conditions.

Rana amurensis and R. dybowskii occupy similar habitats. As temperatures decrease with the onset of winter, both species migrate to ponds for hibernation. Our goal was to determine whether different species possess different intestinal microbiota under natural winter fasting conditions. We used high-throughput Illumina sequencing of 16S rRNA gene sequences to analyse the diversity of intestinal microbes in the two species. The dominant gut bacterial phyla in both species were Bacteroidetes, Proteobacteria and Firmicutes. Linear discriminant analysis (LDA) effect size revealed significant enrichment of Proteobacteria in R. amurensis and Firmicutes in R. dybowskii. There were significant differences in the gut microbiota composition between the species. The core operational taxonomic unit numbers in R. amurensis and R. dybowskii shared by the two species were 106, 100 and 36. This study indicates that the intestinal bacterial communities of the two frog species are clearly different. Phylum-level analysis showed that R. amurensis was more abundant in Proteobacteria and Verrucomicrobia than R. dybowskii was This is the first study of the composition and diversity of the gut microbiota of these two species, providing important insights for future research on the gut microbiota and the role of these bacterial communities in frogs.

Modelling the growth of the brown frog (Rana dybowskii).

Well-controlled development leads to uniform body size and a better growth rate; therefore, the ability to determine the growth rate of frogs and their period of sexual maturity is essential for producing healthy, high-quality descendant frogs. To establish a working model that can best predict the growth performance of frogs, the present study examined the growth of one-year-old and two-year-old brown frogs (Rana dybowskii) from metamorphosis to hibernation (18 weeks) and out-hibernation to hibernation (20 weeks) under the same environmental conditions. Brown frog growth was studied and mathematically modelled using various nonlinear, linear, and polynomial functions. The model input values were statistically evaluated using parameters such as the Akaike's information criterion. The body weight/size ratio (Kwl) and Fulton's condition factor (K) were used to compare the weight and size of groups of frogs during the growth period. The results showed that the third- and fourth-order polynomial models provided the most consistent predictions of body weight for age 1 and age 2 brown frogs, respectively. Both the Gompertz and third-order polynomial models yielded similarly adequate results for the body size of age 1 brown frogs, while the Janoschek model produced a similarly adequate result for the body size of age 2 brown frogs. The Brody and Janoschek models yielded the highest and lowest estimates of asymptotic weight, respectively, for the body weights of all frogs. The Kwl value of all frogs increased from 0.40 to 3.18. The K value of age 1 frogs decreased from 23.81 to 9.45 in the first four weeks. The K value of age 2 frogs remained close to 10. Graphically, a sigmoidal trend was observed for body weight and body size with increasing age. The results of this study will be useful not only for amphibian research but also for frog farming management strategies and decisions.

Combined alkali and acid pretreatment of spent mushroom substrate for reducing sugar and biofertilizer production.

Spent mushroom substrate (SMS) was pretreated with alkaline reagents including potassium hydroxide, lime and ammonia to enhance enzymatic saccharification. Under the best pretreatment conditions (1M KOH, 80 °C, 90 min; 1M lime, 80 °C, 120 min; 10 M ammonia, 70 °C, 120 min), the total reducing sugar (TRS) yield reached 258.6, 204.2 and 251.2 mg/g raw SMS, which were respectively 6.15, 4.86, and 5.98 times of untreated SMS. The effects of pretreatment by above alkaline reagents and sulfuric acid on the composition and structure of SMS were evaluated to provide comparative performance data. A new process, combined alkali and acid (CAA) pretreatment followed by enzymatic hydrolysis, was innovatively proposed to improve the cost-effectiveness and avoid environmental problems. The SMS residue after CAA pretreatment-enzymatic hydrolysis process was converted to biofertilizer with Pichia farinose FL7 and a cell density of 3.0×10(8) cfu/g in biomass was attained.

One-wavelength tuning method for a tunable optical filter based on a cascaded polarization interference filter.

A one-wavelength tuning method is proposed to solve the tuning difficulty in a tunable optical filter based on cascaded cells. This method ensures that the optical path difference variation of all of the cells for the tuning is less than one wavelength and lowers the relative tuning accuracy requirement. Simulations show that different cells have different cross talk and loss deterioration when they undergo an error in optical path difference between the O-ray and the E-ray. An optimal error ratio in each cell is obtained. Under the error ratio, the deterioration from the random error in every cell is further simulated.