Due to its wide distribution across the world, the snail Radix auricularia plays a central role in the transferal of energy and biomass by consuming plant biomass in freshwater systems. The gut microbiota are involved in the nutrition, digestion, immunity, and development of snails, particularly for cellulolytic bacteria, which greatly contribute to the digestion of plant fiber. For the first time, this study characterized the gut bacterial communities of R. auricularia, as well as predicted functions, using the Illumina Miseq platform to sequence 16S rRNA amplicons. Both juvenile snails (JS) and adult snails (AS) were sampled. The obtained 251,072 sequences were rarefied to 214,584 sequences and clustered into 1,196 operational taxonomic units (OTUs) with 97% sequence identity. The predominant phyla were Proteobacteria (JS: 36.0%, AS: 31.6%) and Cyanobacteria (JS: 16.3%, AS: 19.5%), followed by Chloroflexi (JS: 9.7%, AS: 13.1%), Firmicutes (JS: 14.4%, AS: 6.7%), Actinobacteria (JS: 8.2%, AS: 12.6%), and Tenericutes (JS: 7.3%, AS: 6.2%). The phylum Cyanobacteria may have originated from the plant diet instead of the gut microbiome. A total of 52 bacterial families and 55 genera were found with >1% abundance in at least one sample. A large number of species could not be successfully identified, which could indicate the detection of novel ribotypes or result from insufficient availability of snail microbiome data. The core microbiome consisted of 469 OTUs, representing 88.4% of all sequences. Furthermore, the predicted function of bacterial community of R. auricularia performed by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States suggests that functions related to metabolism and environmental information processing were enriched. The abundance of carbohydrate suggests a strong capability of the gut microbiome to digest lignin. Our results indicate an abundance of bacteria in both JS and AS, and thus the bacteria in R. auricularia gut form a promising source for novel enzymes, such as cellulolytic enzymes, that may be useful for biofuel production. Furthermore, searching for xenobiotic biodegradation bacteria may be a further important application of these snails.

The freshwater pulmonate snail Planorbella trivolvis is a common species in various bodies of water but is not native to China. Planorbella trivolvis usually live on diets with high fiber content, such as water grasses, algae and fallen leaves. These snails can attach to the wall of a water tank or to water grass and can be transported overseas to China through the ornamental fish trade. There are few studies investigating the intestinal microbiota of freshwater snails. In this study, using culture-independent molecular analysis, we assessed for the first time the complexity of bacterial communities in the intestines of reared snails. The intestinal microbiota in the snails fed different diets, that is, herbivorous feed (HV) with high cellulose and non-herbivorous feed (NHV) with low cellulose, were analyzed by Illumina sequencing. The results showed that the NHV-based diet significantly increased the body mass, shell diameter and specific growth rate of the snails after 60 days of rearing (P < 0.05). Histological experiments showed that the fat droplets in the epithelium columnar cells of the intestines of the NHV snails increased, and the cilia on these cells fell off. The sequencing results identified 486 and 195 OTUs in HV and NHV, respectively. Lots of bacteria were not reported previously in snails. The intestinal microbiota diversity index (Shannon, Simpson, Ace and Chao) in the NHV snails was significantly lower than that in the HV snails. The gut microbiota in the HV snails were predominantly Proteobacteria (52.97%) and Bacteroidetes (28.75%), while the gut microbiota in NHV snails were predominantly Proteobacteria (95.23%). At the genus level, Cloacibacterium (24.60%), Pseudomonas (4.47%), OM6ON (6.12%), and Rhodobacter (5.79%) were observed to be abundant in HV snails. However, Aeromonas (85.4%) was determined to be predominant in NHV snails. Functional prediction of the gut microbiome in snails by PICRUSt demonstrated a significant difference between the two groups, and the HV snails exhibited higher lignocellulose enzyme activity than did the NHV snails. This study represents a first step in characterizing the gut microbiota of the freshwater snail. Most of these microbes can process plant biomass and digest cellulose and lignocellulose, and the enzymes of these bacteria may have potential biotechnological applications in a variety of industrial processes.

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 (K_wl) 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. TheK_wlvalue of all frogs increased from 0.40 to 3.18. TheKvalue of age 1 frogs decreased from 23.81 to 9.45 in the first four weeks. TheKvalue 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.

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.

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.