Corrigendum: Trophic diversification and parasitic invasion as ecological niche modulators for gut microbiota of whitefish.

[This corrects the article DOI: 10.3389/fmicb.2023.1090899.].

Metapopulation Structure of Two Species of Pikeworm (Triaenophorus, Cestoda) Parasitizing the Postglacial Fish Community in an Oligotrophic Lake.

In the present study, we estimated the levels of infestation of the main fish species that are hosts for two Triaenophorus species: T. crassus and T. nodulosus. The prevalence of T. crassus and T. nodulosus infestations in the intestine of their definitive host-pike Esox lucius was similar (71.0% and 77.4%, respectively). At the same time, the prevalence of T. crassus infestation in muscle tissue was significantly different between the second intermediate hosts, Coregonus lavaretus pidschian (31.4%) and Cor. l. pravdinellus (91.2%), due to considerable differences in their diets. For T. nodulosus, we found significant variations in the levels of prevalence among the second intermediate hosts-100% for Lota lota, 81.8% for Cottus sibiricus 31.9% for Thymallus arcticus, and 24.5% for Perca fluviatilis-that we also explained using different diets. Moreover, analysis of the symmetry of parasite infestations did not reveal any asymmetry between the number of cysts in the left and right body surfaces of the "planktivorous" form/species of whitefish, whereas in the ''benthivorous", an asymmetry of parasite infestations was found.

Trophic diversification and parasitic invasion as ecological niche modulators for gut microbiota of whitefish.

Introduction: The impact of parasites on gut microbiota of the host is well documented, but the role of the relationship between the parasite and the host in the formation of the microbiota is poorly understood. This study has focused on the influence that trophic behavior and resulting parasitism has on the structure of the microbiome. Methods: Using 16S amplicon sequencing and newly developed methodological approaches, we characterize the gut microbiota of the sympatric pair of whitefish Coregonus lavaretus complex and the associated microbiota of cestodes parasitizing their intestine. The essence of the proposed approaches is, firstly, to use the method of successive washes of the microbiota from the cestode's surfaces to analyze the degree of bacterial association to the tegument of the parasite. Secondly, to use a method combining the sampling of intestinal content and mucosa with the washout procedure from the mucosa to understand the real structure of the fish gut microbiota. Results and discussion: Our results demonstrate that additional microbial community in the intestine are formed by the parasitic helminths that caused the restructuring of the microbiota in infected fish compared to those uninfected. Using the desorption method in Ringer's solution, we have demonstrated that Proteocephalus sp. cestodes possess their own microbial community which is put together from "surface" bacteria, and bacteria which are weakly and strongly associated with the tegument, bacteria obtained after treatment of the tegument with detergent, and bacteria obtained after removal of the tegument from the cestodes.

Localization of the proteinase inhibitor activity in the fish cestode Eubothrium rugosum.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.

Seasonal changes in kinetic parameters of trypsin in gastric and agastric fish.

The objective of the present study was to assess if trypsin, a key enzyme involved in protein digestion, presents some kind of functional adaptations to seasonal changes in water temperature in freshwater fish. In order to test this hypothesis, individuals of two fish species Carassius gibelio (agastric) and Perca fluviatilis (gastric) were sampled in the basin of Chany Lake (Siberia, Russia) at two different seasons (spring and summer). Apparent kinetic parameters (Km and Vmax) were determined for both species and seasons at the actual pH values in fish guts, and at actual temperatures. Results showed a significant effect of both the species and sampling season on the apparent kinetic parameters of trypsin. In the case of Prussian carp, Km and Vmax were lower for each assayed temperature (for 5 and 15 °C the differences were significant) for fish sampled in summer when compared to those sampled in spring. In contrast, values of Km in perch tended to be lower in spring at 5 and 25 °C but these differences were not significant, while Vmax showed a significant decrease in summer samples. This suggests a sort of functional adaptation of the same trypsin enzymes to seasonal changes, oriented to maximize protein digestion under variable conditions.

Composition of the microbial communities in the gastrointestinal tract of perch (Perca fluviatilis L. 1758) and cestodes parasitizing the perch digestive tract.

Using the approach of sequencing the V3-V4 region of the 16S rRNA gene, we have analysed the bacterial diversity associated with the distinct compartments of the gastrointestinal tract of perch (Perca fluviatilis) and cestodes (Proteocephalus sp.) parasitizing their digestive tract. The dominant microbiota associated with cestodes (Proteocephalus sp.) was represented by bacteria from the genera Serratia, Pseudomonas and Mycoplasma. By comparing the associated microbiota of perch and cestodes, a clear difference in bacterial composition and diversity was revealed between the community from the stomach content and other parts of the gastrointestinal tract of fish. Microbiota associated with cestodes was not significantly different in comparison with microbiota of different subcompartments of perch (mucosa and content of intestine and pyloric caeca) (ADONIS, p > .05) excluding microbiota of stomach content (ADONIS, p ≤ .05). PICRUSt-based functional assessments of the microbial communities of perch and cestodes indicated that they mainly linked in terms of metabolism and environmental information processing and could play an important role in the nutrition and health of host.

The effect of diet on the structure of gut bacterial community of sympatric pair of whitefishes (Coregonus lavaretus): one story more.

In the Coregonus lavaretus complex may be found lacustrine sympatric pairs, which serves as an intriguing model for studying different aspects of fish evolutionary biology. One such sympatric whitefish pair inhabits Teletskoye Lake (West Siberia, Russia) and includes a "large" form (Coregonus lavaretus pidschian (Gmelin, 1789)) and a "small" form (C. l. pravdinellus (Dulkeit, 1949)). C. l. pravdinellus has a narrow trophic specialization and feeds on zooplankton, whereas the diet of C. l. pidschian is based on benthic prey. In the present study we aimed to address the question of how the gut microbial community reflects the divergence in diet of a sympatric pair of whitefish. Studied samples included the mucosa and content were collected for cardiac and pyloric stomach, anterior, middle, and posterior intestine, but only mucosa was collected for the pyloric caeca. In addition, water, sediment, macrophyte (environmental microbiota) and invertebrate (microbiota of prey) samples were collected in the same location. The V3-V4 region of the 16S rRNA genes was chosen for microbiome analysis and the software PICRUSt used to estimate the difference functional roles of the microbiota. The number of OTUs and Chao1 index in mucosa and content of cardiac and pyloric stomach were significantly different between whitefish. Significant differences were observed between whitefish for content from different parts of the intestine in terms of OTU number and Chao1 indices, whereas for mucosa from the same parts of intestine these differences were absent. No significant differences were found for diversity estimates of mucosa and content of different parts of the gut (there were a few exceptions) between whitefish. The form of whitefish and the segment of the digestive system were factors with a significant determinative effect on the structure of the microbiota from gut mucosa and content. The most dominant phyla in mucosa and content of cardiac and pyloric stomach was Proteobacteria (57.0-84.0%) for both whitefish. Throughout the intestine of C. l. pidschian the dominant phyla in mucosa were Proteobacteria (38.8%) and Firmicutes (15.6%), whereas for C. l. pravdinellus-Tenericutes (49.6%) and Proteobacteria (28.1%). For both forms, the phylum Spirochaetes was found in a significant amount (20.0-25.0%) in the mucosa of the posterior intestine. While for the content obtained from anterior, middle and posterior intestines, the dominant bacterial phyla were the same as those described for mucosa from the same parts of the intestine for both whitefish. The bacterial community of the prey and environment was significantly different from bacterial communities found for all parts of the gut mucosa for both whitefish, with the exception of the mucosa of the cardiac stomach. According to PICRUSt the highest level of differences between whitefish at the L3 level were found for the intestinal mucosa (75.3%), whereas the lowest one was registered for stomach content (38.8%).

DNA extraction protocols may influence biodiversity detected in the intestinal microbiome: a case study from wild Prussian carp, Carassius gibelio.

In this investigation, we examined the influence of different DNA extraction protocols on results obtained for intestinal microbiota of Prussian carp. We showed that significant differences were observed in numbers of reads, OTUs, Shannon index and taxonomic composition between two different DNA extraction protocols for intestine of Prussian carp (Carassius gibelio), and differences were also evident between microbial communities in the intestinal mucosa and intestinal content. Statistical analyses of 25 published articles also revealed a significant relationship between methods of DNA extraction and bacterial diversity in fish intestine of freshwater species. Microbial diversity, community structure, proportions of read numbers derived from each OTU and the total number of OTU's obtained by different DNA extraction protocols could lead to a bias in results obtained in some cases, and therefore researchers should be conservative in conclusions about community structures.

Variations in the activity of digestive enzymes along the intestine of the burbot Lota lota expressed by different methods.

The activities of major digestive hydrolases (proteases, amylase, lipase and esterases) along the intestine were studied in the burbot Lota lota (L.) using different methods of activity expression. The enzyme activities were determined both in the whole gut segments and in the isolated mucosa, and then expressed in terms of tissue mass and protein content in the samples. Further, the cumulative activities of these enzymes in the pyloric caeca were compared with those in the rest of the intestine to estimate the overall contribution of these regions to digestion. The data obtained suggest the essential role of the pyloric caeca in the digestion of the burbot. In addition, the variations in the pH values along the intestine and the changes in the enzyme activities with incubation temperature were examined. The study proved the method of enzyme activity expression to be a key factor influencing the outcome of the experiment.