Phylum Ciliophora Research Articles

Multiple independent genetic code reassignments of the UAG stop codon in phyllopharyngean ciliates.

The translation of nucleotide sequences into amino acid sequences, governed by the genetic code, is one of the most conserved features of molecular biology. The standard genetic code, which uses 61 sense codons to encode one of the 20 standard amino acids and 3 stop codons (UAA, UAG, and UGA) to terminate translation, is used by most extant organisms. The protistan phylum Ciliophora (the 'ciliates') are the most prominent exception to this norm, exhibiting the grfeatest diversity of nuclear genetic code variants and evidence of repeated changes in the code. In this study, we report the discovery of multiple independent genetic code changes within the Phyllopharyngea class of ciliates. By mining publicly available ciliate genome datasets, we discovered that three ciliate species from the TARA Oceans eukaryotic metagenome dataset use the UAG codon to putatively encode leucine. We identified novel suppressor tRNA genes in two of these genomes which are predicted to decode the reassigned UAG codon to leucine. Phylogenomics analysis revealed that these three uncultivated taxa form a monophyletic lineage within the Phyllopharyngea class. Expanding our analysis by reassembling published phyllopharyngean genome datasets led to the discovery that the UAG codon had also been reassigned to putatively code for glutamine in Hartmannula sinica and Trochilia petrani. Phylogenomics analysis suggests that this occurred via two independent genetic code change events. These data demonstrate that the reassigned UAG codons have widespread usage as sense codons within the phyllopharyngean ciliates. Furthermore, we show that the function of UAA is firmly fixed as the preferred stop codon. These findings shed light on the evolvability of the genetic code in understudied microbial eukaryotes.

Comparative genomics provides insights into the evolutionary history of the phylum Ciliophora (Eukaryota, Alveolata) and uncovers the adaptive evolution of anaerobic ciliate classes

Comparative genomics provides insights into the evolutionary history of the phylum Ciliophora (Eukaryota, Alveolata) and uncovers the adaptive evolution of anaerobic ciliate classes

The Phylogenetic Characterization of Balantioides coli Isolated in the Pavlova Culture Medium Supplemented with Coconut Water and Animal Serum.

Balantioides coli is a ciliated protist that can cause dysentery in humans, pigs and nonhuman primates and may have the potential for zoonotic transmission. Its diagnosis is routinely performed through conventional parasitological techniques, and few studies have used culturing techniques to isolate it, applying molecular tools for the characterization of this protozoan. Thus, the objective of this study was to confirm B. coli diagnosis using molecular tools and to characterize the genetic variants of this parasite isolated from pigs kept on family farms in Brazil using three different culture media that differed in the serum added. Fecal samples from pigs were inoculated in Pavlova medium plus coconut water (PC), fetal bovine serum (PB) and horse serum (PH). Of the 127 samples positive for forms compatible with the phylum Ciliophora, 31 were selected for isolation. The most successful medium for isolation was PB 19/31 (61.3%), followed by PH 18/31 (58.1%) and PC 11/31 (35.5%). Of the nucleotide sequences generated, 20 were classified as genetic variant type B0, two as A1 and 15 as A0. The results indicated that PC, despite having allowed the isolation of B. coli for a short period, was not an adequate medium for the maintenance of this parasite in vitro, therefore requiring improvement.

Eukaryotic plankton species diversity and community structure in the Xiao Jiang River (the primary tributary of Upper Yangtze River), Yunnan.

The Xiao Jiang River, as a crucial element of ecological restoration in the upper reaches of the Yangtze River, plays an indispensable role in agricultural water utilization and water ecology within its watersheds. The water quality status of the Xiao Jiang River not only impacts local water-ecological equilibrium and economic benefits but also holds paramount importance for sustaining ecosystem health in the Yangtze River basin. Plankton surveys and environmental physicochemical detection were conducted in the major channel region of the Xiao Jiang River in dry and wet periods in 2022 to better understand the diversity of eukaryotic plankton and its community structure characteristics. Environmental DNA is an emerging method that combines traditional ecology with second-generation sequencing technology. It can detect species from a single sample that are difficult to find by traditional microscopy, making the results of plankton diversity studies more comprehensive. For the first time, environmental DNA was used to investigate eukaryotic plankton in the Xiao Jiang River . The results showed that a total of 881 species of plankton from 592 genera in 17 phyla were observed. During the dry period, 480 species belonging to 384 genera within17 phyla were detected, while, during the wet period, a total of 805 species belonging to 463 genera within 17 phyla were recorded. The phylum Ciliophora dominated the zooplankton, while the phylum Chlorophyta and Bacillariophyta dominated the phytoplankton. The presence of these dominant species indicate that the water quality conditions in the study area are oligotrophic and mesotrophic. Principal coordinate analysis and difference test showed that the number of plankton ASVs, abundance, species richness, dominating species, and diversity indices differed between the dry and wet periods. Spearman correlation analysis and redundancy analysis (RDA) of relative abundance data with environmental physicochemical factors revealed that water temperature (WT), dissolved oxygen (DO), potential of hydrogenacidity (pH), ammonia nitrogen (NH3-N), total nitrogen (TN), electrical conductivity (EC) and the determination of redox potential (ORP) were the main environmental physicochemical factors impacting the plankton community structure. The results of this study can serve as a provide data reference at the plankton level for water pollution management in the Xiao Jiang River, and they are extremely important for river ecological restoration and biodiversity recovery in the Yangtze River basin.

Evaluating nomina of the phylum Ciliophora: examples for increasing work load of serious taxonomists

Thirteen examples mainly taken from ciliatology illustrate the increasing work load of serious taxonomists interested in the reliability of nomenclatural information and trying to be Code-compliant. Weaknesses of the “Amendment” of five articles to expand methods of publication of the Code resulted in the increasing vagueness of dating a nomen (and/or even authorship). The statuses of periodicals with two ISSNs (Print and Online), online-only versions not being or incompletely registered in Zoobank and Corrigenda are often questionable. It is necessary to check in detail the nomenclatural availability of novelties included in them. The Zoobank registration of works published on ciliates is of little help in this respect. There was also retrieval inefficiency and bias of the search engine Global Biodiversity Information Facility (GBIF) tested for a subset of ciliate nomina (nearly 350 nomina and/or spellings) involved in unavailability, mainly objective synonymy and homonymy. The results clearly indicate that the taxonomic status is privileged and nomenclatural revisionary work, which often is spread over three or more decades, is very fragmentarily recognised. Moreover, the main subcategories of synonymy (including alloneonymy) and the correctness of information on senior and junior homonyms are disregarded. Recent monographies are not adequately represented in online databases and websites, forcing genuine taxonomists (after the promulgation of the revisionary work) to check a second time and often to correct each single record in the internet.

Zoonotic Parasites in Artiodactyls with Emphasis on the Feral Boar in the Atlantic Forest, State of Rio de Janeiro, Brazil

Background: The purpose of this study was to identify the species of artiodactyl host related to the fecal matter collected in a forest area in Rio de Janeiro state and carry out a parasitological investigation. Methods: Artiodactyl feces were collected between 2020 and 2021. The fecal samples were examined to identify the host through macroscopic and molecular analysis. The remaining samples were subjected to a fecal parasite analysis using microscopic techniques, and feces containing cysts of the phylum Ciliophora underwent a molecular analysis. Results: Of the 101 samples collected, 71.3% were found in Pavão Valley, the most anthropized area of the park. In the molecular analysis, 79 samples were identified as belonging to Sus scrofa and 2 as Mazama gouazoubira. The most frequently detected forms were cysts of the phylum Ciliophora (39.6%), followed by eggs of Ascaris spp. (11.8%) and Metastrongylus spp. (5.9%). Nucleotide sequences of Balantioides coli were characterized in 26 samples, and in 13 samples variants of type B0 and in 11 type A0 were identified. Conclusions: It should be noted that this is the first study in the Americas that has identified B. coli in free-living S. scrofa feces, citing this bioinvader as one of the reservoirs of this parasite.

Deciphering phylogenetic relationships of and delimiting species boundaries within the controversial ciliate genus Conchophthirus using an integrative morpho-evo approach

The phylum Ciliophora (ciliates) comprises about 2600 symbiotic and over 5500 free-living species. The inclusion of symbiotic ciliates in phylogenetic analyses often challenges traditional classification frameworks due to their morphological adaptions to the symbiotic lifestyle. Conchophthirus is such a controversial obligate endocommensal genus whose affinities to other symbiotic and free-living scuticociliates are still poorly understood. Using uni- and multivariate morphometrics as well as 2D-based molecular and phylogenetic analyses, we attempted to test for the monophyly of Conchophthirus, study the boundaries of Conchophthirus species isolated from various bivalves at mesoscale, and reveal the phylogenetic relationships of Conchophthirus to other scuticociliates. Multidimensional analyses of morphometric and cell geometric data generated the same homogenous clusters, as did phylogenetic analyses based on 144 new sequences of two mitochondrial and five nuclear molecular markers. Conchophthirus is not closely related to ‘core’ scuticociliates represented by the orders Pleuronematida and Philasterida, as assumed in the past using morphological data. Nuclear and mitochondrial markers consistently showed the free-living Dexiotricha and the mouthless endosymbiotic Haptophrya to be the nearest relatives of Conchophthirus. These three highly morphologically and ecologically dissimilar genera represent an orphan clade from the early radiation of scuticociliates in molecular phylogenies.

Multidisciplinary characterization of the new species Copemetopus mystakophoros and its symbionts with a proposal for the new class Copemetopea (Alveolata: Ciliophora)

Abstract The history of the genus Copemetopus is tortuous and studded with several misattributions. It was erected by Villeneuve-Brachon in 1940 after the discovery of Copemetopus subsalsus in saline ponds along the French coast of the Mediterranean Sea near Sète and associated with the class Heterotrichea in the family Metopidae, close to Bryometopus. After a long series of systematic revisions, it is now clear that Copemetopus is not a heterotrich and that it falls in the subphylum Intramacronucleata. Nevertheless, a lot more work is needed to fix the complex taxonomic status of the genus, which lacks a precise taxonomic collocation (it is presently referred to as incertae sedis). In the present study focused on a multidisciplinary and detailed description of a new species, of the genus, Copemetopus mystakophoros sp. nov., we also propose the erection of the new class, Copemetopea cl. nov. After careful literature and data revision, we believe that members of Copemetopus require a higher-ranked taxon in the phylum Ciliophora, given their molecular and morphological peculiarities.

Rediscovery of Remarkably Rare Anaerobic Tentaculiferous Ciliate Genera Legendrea and Dactylochlamys (Ciliophora: Litostomatea).

Free-living anaerobic ciliates are of considerable interest from an ecological and an evolutionary standpoint. Extraordinary tentacle-bearing predatory lineages have evolved independently several times within the phylum Ciliophora, including two rarely encountered anaerobic litostomatean genera, Legendrea and Dactylochlamys. In this study, we significantly extend the morphological and phylogenetic characterization of these two poorly known groups of predatory ciliates. We provide the first phylogenetic analysis of the monotypic genus Dactylochlamys and the three valid species of Legendrea based on the 18S rRNA gene and ITS-28S rRNA gene sequences. Prior to this study, neither group had been studied using silver impregnation methods. We provide the first protargol-stained material and also a unique video material including documentation, for the first time, of the hunting and feeding behavior of a Legendrea species. We briefly discuss the identity of methanogenic archaeal and bacterial endosymbionts of both genera based on 16S rRNA gene sequences, and the importance of citizen science for ciliatology from a historical and contemporary perspective.

A Brief Account on Protozoan Infection in Asian Stinging Catfish, Heteropneustes Fossilis (Bloch, 1794) In Different Seasons of Bangladesh

The study was conducted to identify the protozoan parasites in a freshwater indigenous air breathing fish, Heteropneustes fossilis (Bloch, 1794). The host fish was collected during mid of the April 2018 to end of the March 2019 from freshwater bodies of Mymensingh, Kishoregonj, Faridpur, Jashore, Manikganj and Bogura districts of Bangladesh. Three species of phylum myxozoa namely Henneguya singhi, Henneguya qadrii, Henneguya mystusia, one species of phylum ciliophora namely Trichodina siddiquae and two species of phylum mastigophora namely Trypanosoma singhii and Piscinoodium pillulare were identified in H. fossilis. The parasites of all infected hostswere observed in gill, body slime and blood, however gillswere commonly infected by parasites rather than body slime and blood. Three species of parasite Piscinoodium pillulare, Henneguya qadrii and Henneguya mystusia were first recorded in this host fish and novel locality record in Bangladesh. H. fossilis was found to be infected over the three major (p<0.05) (summer, rainy and winter) seasons and demonstrated a strong significant association (P0<05) with season during the study period. In H. fossilis, highest variety and highest number of parasites were found in winter season, prevalence and intensity was found 78.31% and 5.17 respectively. Lowest prevalence and intensity were found in rainy season respectively 45.87 and 4.72. Among all the parasites Henneguya singhi was common in all the three seasons and its rate of infection was found elevated in summer (20.69%) and rainy season (19.27%). In H. fossilis prevalence of female fish were highest in all the three seasons, summer (55.22%), rainy (50.75%) and winter (85.57%) than their male counterparts 38.76%, 38.09% and 68.12%, respectively. Dhaka Univ. J. Biol. Sci. 32(1): 21-32, 2023 (January)

Ciliate Microtubule Diversities: Insights from the EFBTU3 Tubulin in the Antarctic Ciliate Euplotes focardii.

Protozoans of the Phylum Ciliophora (ciliates) assemble many diverse microtubular structures in a single cell throughout the life cycle, a feature that made them useful models to study microtubule complexity and the role of tubulin isotypes. In the Antarctic ciliate Euplotes focardii we identified five β-tubulin isotypes by genome sequencing, named EFBTU1, EFBTU2, EFBTU3, EFBTU4 and EFBTU5. By using polyclonal antibodies directed against EFBTU2/EFBTU1 and EFBTU3, we show that the former isotypes appear to be involved in the formation of all microtubular structures and are particularly abundant in cilia, whereas the latter specifically localizes at the bases of cilia. By RNA interference (RNAi) technology, we silenced the EFBTU3 gene and provided evidence that this isotype has a relevant role in cilia regeneration upon deciliation and in cell division. These results support the long-standing concept that tubulin isotypes possess functional specificity in building diverse microtubular structures.

Characterization of the complete mitochondrial genome of Miamiensis avidus causing flatfish scuticociliatosis.

Miamiensis avidus is a parasitic pathogen that causes the disease scuticociliatosis in teleost fish species. It is a ciliate and a free-living marine protozoan belonging to the order Philasterida, subclass Scuticociliatida, class Oligohymenophorea, and phylum Ciliophora. The complete mt-genome of M. avidus was linear and 38,695bp in length with 47 genes, including 40 protein-coding genes, two ribosomal RNA (rRNA) genes, and five transfer RNA (tRNA) genes. Of these, 20 genes typically belong to the clusters of orthologous groups, playing roles in energy production and conversion, translation, ribosomal structure and biogenesis, and defense mechanisms. This is the first report of sequencing and characterization of the mt-genome of M. avidus, which was observed to be linear and possessing the typical ciliate mitochondrial genome organization and phylogenetic relationships. Remarkable differences were observed between M. avidus and other ciliates in the mitochondrially encoded rRNAs, extensive gene loss in ribosomal genes and tRNAs, terminal repeat sequences, and stop codon usage. A comparative and phylogenetic analysis of M. avidus and Uronema marinum of the order Hymenostomatida, which is most closely related to the order Philasterida, signified the promise of the mitogenome data of M. avidus as a valuable genetic marker in species detection and taxonomic research. The present study has potential applications in epidemiological studies and host-parasite interaction investigations facilitating disease control.

Report of a protozoan of the phylum Ciliophora compatible with Balantioides coli in feces of white-lipped peccaries in Brazil

Balantioides coli is a ciliated protozoan with worldwide distribution, which parasites several species, especially the pig. In this work, it was presented the first possible case of parasitism by B. coli in white-lipped peccaries in Brazil. The 13 fecal samples were collected from white-lipped peccaries kept in the Zoológico Municipal de Curitiba, Brazil. The feces were processed using flotation and spontaneous sedimentation techniques. All of them were positive for parasites of the phylum Ciliophora similar to B. coli. As the animals did not show clinical signs of balantidiasis, they may be acting as reservoirs, which is worrying because of the proximity of white-lipped peccaries created as a means of subsistence by human population. Thus, we emphasize the importance of documenting these reports in order to understand the expansion of the parasite, mainly due to zoonotic potential of B. coli and implications for public health.

Family-Run Pig Farms: Research and Extension Activities for Parasite Control in a Municipality in the State of Rio de Janeiro, Brazil.

The parasites infesting pigs and pig farmers on family farms in Rio de Janeiro city, Brazil, were analyzed, and extension activities were carried out to impart information about parasites. Between 2020 and 2021, fecal samples were collected from 180 pigs as well as ear scrapings from 142 pigs. In addition, 34 stool specimens from farmers and their families were analyzed. The collected material was processed by parasitological techniques. Parasites were detected in 86.1% of the pigs, forms of phylum Ciliophora (70.5%), strongylids (56.7%), Strongyloides ransomi (44.4%), coccidia (38.3%) and Ascaris suum (32.2%). Sarcoptes scabiei var. suis were identified in 3.5% of the pigs. An analysis of infections by age group revealed that the general frequency of phylum Ciliophora and strongylid parasites was statistically significant. Other factors also associated with the frequency of the parasites included the type of food fed to the pigs, cleaning of the facilities, care of piglets and type of facility. The frequency of intestinal parasites of farmers themselves was low; however, the high rate of parasite infections detected in pigs highlighted the precarious conditions of hygiene of the farms. Lastly, the farmers’ participation in extension activities was a step forward in actions to improve their farm management.

Molecular evolutionary analysis of the SM and SNARE vesicle fusion machinery in ciliates shows concurrent expansions in late secretory machinery.

Protists in the phylum Ciliophora possess a complex membrane-trafficking system, including osmoregulatory Contractile Vacuoles and specialized secretory organelles. Molecular cell biological investigations in Tetrahymena thermophila have identified components of the protein machinery associated with the secretory organelles, mucocysts. The Qa-SNARE Syn7lp plays a role in mucocyst biogenesis as do subunits of the CORVET tethering complex (specifically Vps8). Indeed, Tetrahymena thermophila possesses expanded gene complements of several CORVET components, including Vps33 which is also a Sec1/Munc18 (SM) protein that binds Qa-SNAREs. Moreover, the Qa-SNAREs in Paramecium tetraurelia have been localized to various endomembrane organelles. Here, we use comparative genomics and phylogenetics to determine the evolutionary history of the SM and Qa-SNARE proteins across the Ciliophora. We identify that the last ciliate common ancestor possessed the four SM proteins and six Qa-SNAREs common to eukaryotes, including the uncommonly retained Syntaxin 17. We furthermore identify independent expansion of these protein families in several ciliate classes, including concurrent expansions of the SM protein-Qa SNARE partners Sec1:SynPM in the oligohymenophorean ciliates lineage, consistent with novel Contractile Vacuole specific innovations. Overall, these data are consistent with SM proteins and Qa-SNAREs being a common set of components for endomembrane modulation in the ciliates.

Crustacean and protozoan parasites of some cyprinid fish living in the Murat River (Bingöl-Türkiye), with new host records

Some Cyprinid fish species: Cyprinion macrostomum (Heckel, 1843), Capoeta umbla (Heckel, 1843), Chondrostoma regium (Heckel, 1843), and Squalius cephalus (Linnaeus, 1758) living naturally in the Murat River, were investigated for Protozoan and Crustacean parasite fauna and their distribution. Fish samples were collected from different stations between July 2017 - June 2019, examined in the Bingöl University Zoology Research Laboratory, and the data were explained with various variables. The normality test revealed that the data were not normally distributed (p< 0.05), as with large samples, so non-parametric tests explained the data. A total of 365 fish were examined, and 100 fish (27.4%) were infected with at least one Protozoan or Crustacean parasite. Four different parasite species were recorded on the examined fish, namely Ichthyophthirius multifiliis and Trichodina sp. belonging to the phylum Ciliophora (Protozoan), Ergasilus sieboldi, and Lamproglena pulchella belonging to the phylum Arthropoda (Crustacean). As a result of this study, for the first time, Protozoan and Crustacean parasites of different cyprinid fish were examined according to the host species, seasonal distribution, host size, and new host records were reported for three parasites.

Taxonomical Accounts of Protozoan Parasites of Climbing Perch, Anabas Testudineus in Bangladesh

The study was conducted to identify the protozoan parasites in a freshwater indigenous air breathing fish, Anabas testudineus. The host fish was collected during mid of the April 2018 to end of the March 2019 from freshwater bodies of Mymensingh, Kishoregonj, Faridpur, Jashore, Manikganj and Bogura districts of Bangladesh. Five species of phylum myxozoa namely Henneguya gigas, Henneguya mystusia, Henneguyaa cerinae, Henneguya periophthalmusi and Myxidium sp., three species of phylum ciliophora namely Amphileptus disciformis, Epistylisl woffi and Trichodina anabasi and one species of phylum mastigophora namely Trypanosoma anabasi were identified in A. testudineus. Myxozoans (97.55%) were clearly dominant group than chiliophorans (2.45%). Bangladesh J. Zool. 49 (2): 331-345, 2021

Komunitas makrozoobentos dan kaitannya dengan kualitas air aliran sungai Senggarang, Kota Tanjungpinang

Senggarang is one of the coastal villages in Tanjungpinang City which has a river that flows from the upstream downstream into the sea. The flow of the Senggarang river has distinctive characteristics that are commonly found in small island waters, namely having a relatively short upstream to downstream distance, and the downstream part is influenced by tidal conditions. Various activities along the Senggarang river flow would cause changes in water quality which would impact the presence of macrozoobenthic. The objective of this study was to determine the relationship between the macrozoobenthic community and the water quality of the Senggarang river. Macrozoobenthic sampling, and environmental parameters were carried out using random sampling methods. The results of this study were obtained 27 species from 6 phylum, namely phylum Arthropoda 13 species, phylum Ciliophora 3 species, phylum Echinodermata 1 species, phylum Mollusca 4 species, phylum Nematoda 5 species, and phylum Tardigrada 1 species. The average macrozoobenthic density is 172 ind./ m3 for freshwater and 704 ind./ m3 for brackish waters. The average diversity value for freshwater is 1.29 and 2.16 waters with medium salinity, the average uniformity value of 0.85 freshwater and 0.94 of high in brackish waters, the average dominance value of 0.32 freshwater and 0.13 of low in brackish waters. The relationship of macrozoobenthos with the quality of freshwater based on PCA analyses related to temperature and dissolved oxygen, in brackish waters to temperature, dissolved oxygen, and current.Keywords: Macrozoobenthic community, PCA, River flow, Water quality

Abundance of anuran parasites and pesticide pollution from lowland and highland golf courses

Aim: This study aimed to determine the pesticide pollution and prevalence of protozoa in anurans from lowland and highland golf courses in Malaysia and to observe the relationship between environmental factors (water quality index and pollutants) and the presence of protozoa in anurans. Methodology: Water quality index and pesticide levels (metsulfuron methyl) were measured at three locations, i.e. Jeriau river, Fraser's hill golf course in Pahang, and UKM Danau golf club in Selangor. Microscopy techniques were used to identify and examine the prevalence of protozoa and haemoparasites in anuran organs (stomach, kidney, liver, intestine, rectum, and blood). Results: A total of 140 anurans were collected from the study areas. Jeriau river had the highest water quality compared to Fraser's hill golf course and UKM Danau golf club. Metsulfuron methyl concentrations at all three locations were under permissible level (0.001–0.01 mg l-1). Approximately, 30% of the anurans were infected with ≥ 1 protozoa, of which 22.14% were from phylum Opalinata and 10.71% were from phylum Ciliophora (Nyctotherus cordiformis). Approximately 44.44% of the anurans were infected with ≥ 1 groups of haemoparasites, which included Trypanosoma, Haemogregarina, Lankesterella, Aegyptianella and Microfilaria. Interpretation: No significant relationship was found between the environmental parameters and the abundance of protozoa and haemoparasites in anurans from three locations, indicating that the prevalence of protozoa and haemoparasites in anurans is not influenced by environmental factors.

Active Rumen Bacterial and Protozoal Communities Revealed by RNA-Based Amplicon Sequencing on Dairy Cows Fed Different Diets at Three Physiological Stages.

Seven Italian Simmental cows were monitored during three different physiological stages, namely late lactation (LL), dry period (DP), and postpartum (PP), to evaluate modifications in their metabolically-active rumen bacterial and protozoal communities using the RNA-based amplicon sequencing method. The bacterial community was dominated by seven phyla: Proteobacteria, Bacteroidetes, Firmicutes, Spirochaetes, Fibrobacteres, Verrucomicrobia, and Tenericutes. The relative abundance of the phylum Proteobacteria decreased from 47.60 to 28.15% from LL to DP and then increased to 33.24% in PP. An opposite pattern in LL, DP, and PP stages was observed for phyla Verrucomicrobia (from 0.96 to 4.30 to 1.69%), Elusimicrobia (from 0.32 to 2.84 to 0.25%), and SR1 (from 0.50 to 2.08 to 0.79%). The relative abundance of families Succinivibrionaceae and Prevotellaceae decreased in the DP, while Ruminococcaceae increased. Bacterial genera Prevotella and Treponema were least abundant in the DP as compared to LL and PP, while Ruminobacter and Succinimonas were most abundant in the DP. The rumen eukaryotic community was dominated by protozoal phylum Ciliophora, which showed a significant decrease in relative abundance from 97.6 to 93.9 to 92.6 in LL, DP, and PP, respectively. In conclusion, the physiological stage-dependent dietary changes resulted in a clear shift in metabolically-active rumen microbial communities.