Coleps shanghaiensis n. sp. challenges the validity of the genus Levicoleps (Cilophora, Prostomatida).

Coleps is a common genus of pelagic ciliates in freshwater and brackish water habitats. Classification and phylogeny of Coleps species are, however, still full of confusion. In this study, we investigated Coleps shanghaiensis n. sp., collected from a river in Shanghai, China, by living observation, protargol staining, and molecular methods. Coleps shanghaiensis is about 70-90 µm × 35-55 µm in size, has a barrel-shaped body with three posterior spines, and possesses 21-24 ciliary rows, each composed of two perioral dikinetids and 19-22 monokinetids, and six caudal cilia. In SSU rRNA gene phylogenies, C. shanghaiensis fell within the clade of subspecies of Levicoleps biwae, which questions the validity of the genus Levicoleps. Furthermore, the biogeography of the genus Coleps is discussed.

A new contribution to the raptorial ciliate genus Lacrymaria (Protista: Ciliophora): a brief review and comprehensive descriptions of two new species from Changjiang Estuary.

Ciliates serve as excellent indicators for water quality monitoring. However, their utilization is hindered by various taxonomic confusions. The ciliate genus Lacrymaria Bory de Saint-Vincent, 1824 is commonly found in different aquatic habitats, but its taxonomy has been sparsely investigated using state-of-the-art methods. This study investigated two new Lacrymaria species from Nanhui Wetland, Shanghai, China, using living observation, protargol staining, and molecular phylogeny methods. Lacrymaria songi sp. nov. is 180-340 × 20-25 µm in size and possesses 12-16 somatic kineties, 1 terminal contractile vacuole, 2 macronuclear nodules, and 2 types of rod-shaped extrusomes. Lacrymaria dragescoi sp. nov. is distinguished from its congeners by its cell size of 210-400 × 25-35 µm, 14-17 somatic kineties, 1 terminal contractile vacuole, 1 macronucleus, and 2 types of rod-shaped extrusomes. Phylogenetic analyses based on SSU rRNA gene sequences indicate that Lacrymariidae is monophyletic but Lacrymaria is not. Additionally, a brief review of the genus Lacrymaria is provided in this study. We suggest that L. bulbosa Alekperov, 1984, L. lanceolata Kahl, 1930, and L. ovata Burkovsky, 1970 be removed from the genus and propose Phialina lanceolata nov. comb. and Phialina ovata nov. comb. for the latter two. ZooBank registration: Present work: urn:lsid:zoobank.org:pub:CDFB1EBD-80BD-4533-B391-CEE89F62EDC4 Lacrymaria songi sp. nov.: urn:lsid:zoobank.org:act:417E7C2D-DAEC-4711-90BB-64AB3CD2F7D5 Lacrymaria dragescoi sp. nov.: urn:lsid:zoobank.org:act:8778D6B0-1F2E-473C-BE19-3F685391A40D.

Development and characterization of a spleen cell line from yellowfin seabream Acanthopagrus latus and its susceptibility to Mandarinfish ranavirus.

Yellowfin seabream (Acanthopagrus latus) is one of the most commercially important marine fish in China. In this study, a new continuous cell line, named ALS cells, was developed from the spleen tissue of A. latus. The cell line was maintained in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and successfully cultured up to 50 passages. The cell line was authenticated by amplifying and sequencing mitochondrial cytochrome C oxidase subunit-I (coi-I) gene. The ALS cell line had the maximum growth rate in DMEM/F-12 medium containing 20% FBS at 27°C. Chromosome number analysis showed that the ALS cells have a modal diploid chromosome number of 34. The ALS cell line was transfected with the pEGFP-N1 plasmid, and green fluorescence was observed. The ALS cell line was used for testing Mandarinfish ranavirus (MRV) susceptibility, and the cytopathic effects in the cell line were observed at 4 days post-infection (dpi). Furthermore, the susceptibility of the ALS cell line to MRV and the levels of MRV mRNA and viral loads were found to be significantly increased at 1-7 dpi. This study revealed that the ALS cell line could be useful for molecular, virological, and biotechnological studies on yellowfin seabream.

Taxonomy and molecular phylogeny of pleurostomatid ciliates from China with a description of two new species.

Ciliates in the order Pleurostomatida are found free-living in many habitats including within biofilms, but some (e.g. Pseudoamphileptus spp.) are ectocommensal on various hosts. Due to issues involving overall undersampling, the exact diversity and molecular phylogeny of this group remain largely underexplored. To combat this deficiency, detailed investigations were undertaken in northern China. As a result of these studies, we provide the morphological descriptions of two new species. Pseudoamphileptus apomacrostoma sp. nov., a new ectocommensal species, is characterized by the broadly oval cell shape, numerous scattered contractile vacuoles, and unique densely bounded extrusomes; Amphileptus qingdaoensis sp. nov., a marine form, is characterized by possessing oblong extrusomes with a conical anterior end, a single contractile vacuole and 5-7 left and 18-23 right kineties. In addition, a new population of Amphileptus orientalis Zhang et al., 2022, a freshwater representative, was documented and an improved diagnosis is provided. The phylogenetic analyses based on the SSU rDNA sequences imply that the genus Pseudoamphileptus is monophyletic whereas the genus Amphileptus is paraphyletic. The new molecular sequences presented here further support the establishment of two new species.

Identification through machine learning of potential immune- related gene biomarkers associated with immune cell infiltration in myocardial infarction.

BACKGROUND: To investigate the potential role of immune-related genes (IRGs) and immune cells in myocardial infarction (MI) and establish a nomogram model for diagnosing myocardial infarction. METHODS: Raw and processed gene expression profiling datasets were archived from the Gene Expression Omnibus (GEO) database. Differentially expressed immune-related genes (DIRGs), which were screened out by four machine learning algorithms-partial least squares (PLS), random forest model (RF), k-nearest neighbor (KNN), and support vector machine model (SVM) were used in the diagnosis of MI. RESULTS: The six key DIRGs (PTGER2, LGR6, IL17B, IL13RA1, CCL4, and ADM) were identified by the intersection of the minimal root mean square error (RMSE) of four machine learning algorithms, which were screened out to establish the nomogram model to predict the incidence of MI by using the rms package. The nomogram model exhibited the highest predictive accuracy and better potential clinical utility. The relative distribution of 22 types of immune cells was evaluated using cell type identification, which was done by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm. The distribution of four types of immune cells, such as plasma cells, T cells follicular helper, Mast cells resting, and neutrophils, was significantly upregulated in MI, while five types of immune cell dispersion, T cells CD4 naive, macrophages M1, macrophages M2, dendritic cells resting, and mast cells activated in MI patients, were significantly downregulated in MI. CONCLUSION: This study demonstrated that IRGs were correlated with MI, suggesting that immune cells may be potential therapeutic targets of immunotherapy in MI.

New contributions to the Cyrtophoria ciliates (Protista, Ciliophora): Establishment of new taxa and phylogenetic analyses using two ribosomal genes.

Periphytic ciliates play a vital role in the material cycle and energy flow of microbial food web, however, their taxonomy and biodiversity are inadequately studied given their high species richness. Two new and one little known species, viz. Derouxella lembodes gen. et sp. nov., Cyrtophoron multivacuolatum sp. nov., and Cyrtophoron apsheronica Aliev, 1991, collected from coastal waters of China, were investigated using modern methods. Derouxella gen. nov. can be recognized by having dorsoventrally flattened body, a podite, one fragmented preoral kinety, two parallel circumoral kineties, and somatic kineties progressively shortened from right to left. Morphological classification and phylogenetic analyses based on nuclear small subunit ribosomal RNA (nSSU rRNA) and mitochondrial small subunit ribosomal RNA (mtSSU rRNA) gene sequence data inferred that Derouxella gen. nov. occupies an intermediate position between Hartmannulidae and Dysteriidae. Cyrtophoron multivacuolatum sp. nov. is characterized by large body size, the numbers of somatic kineties and nematodesmal rods, and having numerous contractile vacuoles. The genus Cyrtophoron and the poorly known species C. apsheronica were redefined. Even with the addition of newly obtained nSSU rRNA and mtSSU rRNA gene sequences of Cyrtophoron, the family Chlamydodontidae was still recovered as a monophyletic group, the monophyly of Cyrtophoron was supported too.

Two hypotrichs (Ciliophora, Hypotricha) from China: morphology and SSU rDNA sequence of Holosticha aestuarina nov. spec. and H. muuiensis Kim et al., 2017.

The morphology, four ontogenetic stages, and the phylogenetic relationships based on the small subunit RNA-gene of Holosticha aestuarina nov. spec. from the Yangtze River Estuary (Shanghai) were investigated. The new species differs from the congeners by the following combination of features: body size 160-196 × 27-36 µm in vivo, a vesicular structure containing one or two bean-shaped opaque particles, 24-32 adoral membranelles, 16-34 midventral pairs, 17-26 left marginal cirri, 22-32 right marginal cirri, six or seven transverse cirri, four dorsal kineties, and usually two macronuclear nodules. The anlage for the left marginal row of the proter very likely originates de novo (anterior portion) and from parental cirri (posterior portion). Further, we provide a morphological description of a Chinese population of H. muuiensis Kim et al., 2017, originally discovered in South Korea, indicating that it is confined to the Eastern Asia region. Its SSU rDNA sequence is identical with that of the type population and forms a clade with the available Holosticha sequences. By contrast, H. aestuarina nov. spec. is sister to the Holosticha + Uncinata clade, indicating that Uncinata is a subgenus of Holosticha.

Forensic Research Progress on Postural Asphyxia.

In recent years, the increase in the number of cases of postural asphyxia has gradually attracted the attention and discussion of forensic scientists domestically and internationally, but a systematic, comprehensive and recognized expert consensus and identification standard has not been established at home and abroad. This paper reviews the case characteristics, occurrence, mechanism of death, and identification criteria of postural asphyxia, to provide reference for future research.

Historical Review of Studies on Cyrtophorian Ciliates (Ciliophora, Cyrtophoria) from China.

The subclass Cyrtophoria are a group of morphologically specialized ciliates which mainly inhabit soil, freshwater, brackish water, and marine environments. In this study, we revise more than 50 publications on the taxonomy, phylogeny, and ecology of cyrtophorian ciliates in China since the first publication in 1925, most of which were carried out in coastal areas. The research history can be divided into three periods: the early stage, the Tibet stage, and the molecular stage. To date, 103 morpho-species (147 isolates) have been formally recorded in China, with ciliature patterns described for 82 of them. A species checklist and an illustrated identification key to the genera are provided. A total of 100 small subunit rDNA sequences have been obtained for 74 taxonomic hits (lowest taxonomic rank to species or genus). These sequences are used for the study of molecular phylogeny. Based on these morphological data and molecular phylogeny analyses, we synthesize the understanding of the phylogeny of cyrtophorian ciliates. We hypothesize that the key evolutionary event of cyrtophorian ciliates lies in the separation of the stomatogenesis zone (postoral kineties) from the left kineties, namely, the formation of an independent "sexual organelle". We, furthermore, briefly summarize the ecological features of cyrtophorian ciliates and provide a comprehensive bibliography of related research from China. Finally, we give an outlook on the future research directions of these taxa.

The Capsid Protein of Nervous Necrosis Virus Antagonizes Host Type I IFN Production by a Dual Strategy to Negatively Regulate Retinoic Acid-Inducible Gene-I-like Receptor Pathways.

Nervous necrosis virus (NNV), a highly pathogenic RNA virus, is a major pathogen in the global aquaculture industry. To efficiently infect fish, NNV must evade or subvert the host IFN for their replication; however, the precise mechanisms remain to be elucidated. In this study, we reported that capsid protein (CP) of red-spotted grouper NNV (RGNNV) suppressed the IFN antiviral response to promote RGNNV replication in Lateolabrax japonicus brain cells, which depended on the ARM, S, and P domains of CP. CP showed an indirect or direct association with the key components of retinoic acid-inducible gene-I-like receptors signaling, L. japonicus TNFR-associated factor 3 (LjTRAF3) and IFN regulatory factor (LjIRF3), respectively, and degraded LjTRAF3 and LjIRF3 through the ubiquitin-proteasome pathway in HEK293T cells. Furthermore, we found that CP potentiated LjTRAF3 K48 ubiquitination degradation in a L. japonicus ring finger protein 114-dependent manner. LjIRF3 interacted with CP through the S domain of CP and the transcriptional activation domain or regulatory domain of LjIRF3. CP promoted LjIRF3 K48 ubiquitination degradation, leading to the reduced phosphorylation level and nuclear translocation of LjIRF3. Taken together, we demonstrated that CP inhibited type I IFN response by a dual strategy to potentiate the ubiquitination degradation of LjTRAF3 and LjIRF3. This study reveals a novel mechanism of RGNNV evading host immune response via its CP protein that will provide insights into the complex pathogenesis of NNV.

Clinical assessment and molecular mechanism of the upregulation of Toll-like receptor 2 (TLR2) in myocardial infarction.

OBJECTIVE: The prevalence and mortality of cardiovascular diseases remain ranked first worldwide. Myocardial infarction (MI) is the central cause of death from cardiovascular diseases, seriously endangering human health. The clinical implication of toll-like receptor 2 (TLR2) remains contradictory, and its mechanism is still unknown. Hence, the objective of this study was to elucidate the clinical value and molecular mechanism of TLR2 in MI. METHODS: All high-throughput datasets and eligible literature were screened, and the expression levels of TLR2 were collected from the MI. The integrated expression level of TLR2 was displayed by calculating the standardized mean difference (SMD) and the area under the curve (AUC) of the summary receiver operating characteristic curve (sROC). The related TLR2 genes were sent for pathway analyses by gene ontology (GO), Kyoto encyclopedia of genes and genome (KEGG), and disease ontology (DO). Single-cell RNA-seq was applied to ascertain the molecular mechanism of TLR2 in MI. RESULTS: Nine microarrays and four reported data were available to calculate the comprehensive expression level of TLR2 in MI, including 325 cases of MI and 306 cases of controls. The SMD was 2.55 (95% CI = 1.35-3.75), and the AUC was 0.76 (95% CI = 0.72-0.79), indicating the upregulation of TLR2 in MI. The related TLR2 genes were primarily enriched in the pathways of atherosclerosis, arteriosclerotic cardiovascular disease, and arteriosclerosis, suggesting the clinical role of TLR2 in the progression of MI. Afterward, TLR2 was upregulated in myeloid cells in MI. CONCLUSIONS: TLR2 may have a crucial role in progressing from coronary atherosclerosis to MI. The upregulation of TLR2 may have a favorable screening value for MI.

Taxonomy and SSU rRNA gene-based phylogeny of two new Euplotes species from China: E. chongmingensis n. sp. and E. paramieti n. sp. (Protista, Ciliophora).

BACKGROUND: The genus Euplotes Ehrenberg, 1830, one of the most complicated and confused taxa, contains about 160 nominal species. It was once proposed to be divided into four genera, two of which were proved to be non-monophyletic. At least 19 new species have been discovered in the past decade, implying that there is a large undiscovered diversity of this genus. RESULTS: The morphology of two new freshwater euplotid ciliates, Euplotes chongmingensis n. sp. and E. paramieti n. sp., isolated from Shanghai, China, were investigated using live observations, protargol staining, and Chatton-Lwoff silver staining method. Euplotes chongmingensis is characterized by its small size (40-50 × 25-35 µm), about 24 adoral membranelles, 10 frontoventral cirri, two marginal and two caudal cirri, eight dorsolateral kineties with 11-16 dikinetids in the mid-dorsolateral kinety and a double type of silverline system. Euplotes paramieti n. sp. is 180-220 × 110-155 µm in vivo and strongly resembles E. amieti but having a difference of 57 bp in their SSU rRNA gene sequences. Phylogenetic analyses based on SSU rRNA gene sequence data were used to determine the systematic positions of these new taxa. CONCLUSIONS: The description of two new freshwater taxa and their SSU rRNA gene sequences improve knowledge of biodiversity and enrich the database of euplotids. Furthermore, it offers a reliable reference for environmental monitoring and resource investigations.

Photocatalytic Removal of Cr(VI) by Thiourea Modified Sodium Alginate/Biochar Composite Gel.

Heavy metal pollution is an important problem in current water treatments. Traditional methods for treating chromium-containing wastewater have limitations such as having complicated processes and causing secondary pollution. Therefore, seeking efficient and fast processing methods is an important research topic at present. Photocatalysis is an efficient method to remove Cr(VI) from aqueous solutions; however, conventional photocatalysts suffer from a low metal absorption capacity, high investment cost, and slow desorption of trivalent chromium from the catalyst surface. In this study, a novel composite gel was synthesized by chemically modifying thiourea onto sodium alginate, which was then mixed with biochar. The composite gel (T-BSA) can effectively remove 99.98% of Cr(VI) in aqueous solution through synergistic adsorption and photocatalytic reduction under UV light irradiation. The removal mechanism of Cr(VI) was analyzed by FT-IR, FESEM, UV-DRS and XPS. The results show that under acidic conditions, the amino group introduced by chemical modification can be protonated to adsorb Cr(VI) through electrostatic interaction. In addition, the biochar as a functional material has a large specific surface area and pore structure, which can provide active sites for the adsorption of Cr(VI), while the photo-reduced Cr(III) is released into the solution through electrostatic repulsion, regenerating the adsorption sites, thereby improving the removal performance of Cr(VI). Biochar significantly intensifies the Cr(VI) removal performance by providing a porous structure and transferring electrons during photoreduction. This study demonstrates that polysaccharide-derived materials can serve as efficient photocatalysts for wastewater treatment.

Taxonomy and phylogeny of the freshwater tintinnid Tintinnopsis tubuformis Chiang, 1956 (Ciliophora, Oligotrichea) and a proposed synonymization of T. longa nom. corr. Chiang, 1956.

Tintinnid ciliates are traditionally identified by their loricae; however, increasing evidence indicates that some lorica features (e.g. its length, spiraled structures) are not reliable. The vast majority of tintinnids inhabit the marine pelagial; merely, about thirty species live in freshwater. In the present study, two morphotypes with similar lorica shapes and opening diameters but deviating lorica lengths were isolated from freshwater samples collected at different water temperatures near Chongming Island in the Yangtze Estuary, China. The specimens were studied in vivo and after protargol staining, and their phylogenetic placement was inferred from three ribosomal RNA markers; further, cell division was investigated in the short morphotype. Based on the original descriptions, the longer morphotype is identified as Tintinnopsis longa nom. corr. Chiang, 1956, and the shorter one as Tintinnopsis tubuformis Chiang, 1956. Despite distinct differences in the lorica lengths, the identity of the three molecular markers in both morphotypes suggests conspecificity, which is supported by overlapping ranges in the lorica opening diameters and the length-independent features of the somatic ciliary pattern (e.g. number of kineties). Hence, we synonymized T. longa nom. corr. with T. tubuformis and neotypified the later species.

Re-description and molecular phylogeny of the free-swimming peritrichs Hastatella radians Erlanger, 1890 and H. aesculacantha Jarocki & Jakubowska, 1927 (Ciliophora, Peritrichia) from China.

The present work investigated Hastatella radians Erlanger, 1890 (type species), and H. aesculacantha Jarocki & Jakubowska, 1927, isolated from Chongming Island, Shanghai, China, based on light microscopy observations of live individuals, silver staining and molecular phylogeny methods. Hastatella radians is characterized by its size (about 30-75 µm long, 25-70 µm wide), two girdles of ectoplasmic processes, infundibular polykinety 3 (P3) composed of two rows of unequal length and terminating beyond adstomal end of P2, 8-22 transverse silverlines between peristome and trochal band, and 5-9 between trochal band and scopula. The infraciliature of H. aesculacantha is described for the first time, and we improved its diagnosis: cell size about 25-52 × 20-41 µm in vivo; four girdles of ectoplasmic processes; P3 two-rowed, outer row much shorter than inner one; 5-7 silverlines between peristome and trochal band and four or five between trochal band and scopula. Phylogenetic analyses based on SSU rDNA sequences revealed that Hastatella is monophyletic and has a close relationship to Astylozoon enriquesi.

Integrative studies on three new freshwater Amphileptus species (Ciliophora, Pleurostomatida) discovered in northern China.

The morphology and molecular phylogeny of freshwater pleurostomatid ciliates are insufficiently explored. In the present study, we investigated three new Amphileptus species discovered in Lake Weishan and its vicinity, northern China, using standard alpha-taxonomic methods. Amphileptus paracarchesii sp. nov. is characterized by a lateral fossa (groove) in the posterior body portion, four macronuclear nodules, contractile vacuoles distributed along the dorsal margin, and 4-6 left and 44-50 right somatic kineties. Amphileptus pilosus sp. nov. differs from congeners by having 4-14 macronuclear nodules, numerous contractile vacuoles scattered throughout the cytoplasm, and 22-31 left and 35-42 right somatic kineties. Amphileptus orientalis sp. nov. is characterized by two ellipsoidal macronuclear nodules, three ventral contractile vacuoles, and about four left and 31-35 right somatic kineties. Phylogenetic analyses of nuclear small subunit ribosomal DNA (SSU rDNA) sequences indicate that the family Amphileptidae might be monophyletic while the genus Amphileptus is paraphyletic, as Pseudoamphileptus macrostoma robustly groups with Amphileptus sp. Although deep phylogenetic relationships of amphileptids are poorly resolved, multiple well-delimited species groups are recognizable within the genus Amphileptus. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00143-0.

Taxonomic and phylogenetic studies on two new freshwater Amphileptus species (Ciliophora, Pleurostomatida), from Lake Weishan, northern China.

The morphology and phylogeny of two new species of pleurostomatid ciliates, Amphilepus weishanensis sp. nov. and A. parapleurosigma sp. nov., collected from Lake Weishan in northern China, were studied using live observation, protargol staining and phylogenetic analyses based on small subunit ribosomal DNA (SSU rDNA) sequence data. In both species, extrusomes form an apical group and contractile vacuoles are distributed along both the dorsal and the ventral margin. Amphileptus weishanensis sp. nov. is characterized by its body size (560-780 × 60-100 µm in vivo) and the possession of five left and 56-61 right somatic kineties, filiform extrusomes and 3-9 macronuclear nodules. Amphileptus parapleurosigma sp. nov. is characterized by possessing 4-6 left and 19-24 right somatic kineties and clavate extrusomes. The SSU rDNA sequences differ among ten similar species by 3-60 bp. Phylogenetic analyses based on the SSU rDNA sequences indicate that the family Amphileptidae is monophyletic while Amphileptus is paraphyletic with Pseudoamphileptus nesting within this genus.

MicroRNA expression profiling of sea perch brain cells reveals the roles of microRNAs in autophagy induced by RGNNV infection.

Nervous necrosis virus (NNV) is one of the most destructive fish viruses and affects more than 120 marine and freshwater teleost species. However, the pathogenesis of NNV has not been made clear. MicroRNAs (miRNAs) play important roles in the regulation of viral infection. To understand the roles and regulation patterns of miRNAs in NNV infection, high-throughput sequencing was carried out in Lateolabrax japonicus brain (LJB) cells with or without red-spotted grouper NNV (RGNNV) infection at 12 and 24 hr. Here, we identified 59 known and 61 novel differentially expressed miRNAs (DE miRNAs) between mock and RGNNV-infected LJB cells. KEGG pathway analysis showed that the target genes of DE miRNAs were significantly enriched in immune-related signalling pathways, such as autophagy, mitophagy and TGF-beta signalling pathways. The expression patterns of four DE miRNAs (lja-miR-145, lja-miR-182, lja-miR-183 and lja-miR-187) were verified by qRT-PCR both in vivo and in vitro. We found that lja-miR-145 promoted RGNNV proliferation, while lja-miR-183 suppressed RGNNV proliferation. Furthermore, lja-miR-145 facilitated RGNNV-induced autophagy activation, whereas lja-miR-183 repressed autophagy in LJB cells as measured by LC3B-II/I and p62 protein levels. All these results indicate the involvement of lja-miR-145 and lja-miR-183 in RGNNV-induced autophagy. In conclusion, this study provides evidence for the important roles of miRNAs in NNV infection and a basis for uncovering the molecular regulation mechanism of NNV-induced autophagy.

Morphology and Phylogeny of Two Novel Pleurostomatids (Ciliophora, Litostomatea), Establishing a New Genus.

Pleurostomatida Schewiakoff, 1896 is a cosmopolitan order of ciliates. In the present study, we investigated two new pleurostomatid species, Apolitonotus lynni gen. et sp. nov. and Protolitonotus clampi sp. nov., with state-of-the-art methods. Apolitonotus lynni lacks its oral extrusomes and its right kineties form an anterior semi-suture near the dorsal margin. Based on these two features, the new genus Apolitonotus was established within the Protolitonotidae Wu et al., 2017. Protolitonotus clampi differs from its congeners by its size of 80-130 × 15-30 µm, 4-6 left, and 9-11 right kineties, extrusomes arranged along the oral slit, and two macronuclear nodules. Because Litonotus antarcticus possesses an anterior semi-suture and oral extrusomes, it was transferred to the genus Protolitonotus, becoming P. antarctius comb. nov. (basionym Litonotus antarcticus Song and Wilbert, 2002). Phylogenetic analyses based on SSU rDNA sequences suggest a sister group relationship of P. clampi and the family Kentrophyllidae, and A. lynni is adelphotaxon to Litonotus gracilis, both within the order Pleurostomatida. Based on the new findings, an improved diagnosis for Protolitonotus was also provided.

Towards an eDNA metabarcode-based performance indicator for full-scale municipal wastewater treatment plants.

Knowledge-driven management for wastewater treatment plant (WWTP) performance becomes increasingly important considering the globally growing production of wastewater and the rising demand of clean water supply. Even though the potential of microbial organisms (bacteria and protists) as bioindicators for WWTP performance is well known, it is far from being fully exploited for routine monitoring programs. Therefore, we here used massive sequencing of environmental (e)DNA metabarcodes from bacterial (V3-V4 region of the SSU rRNA gene) and eukaryote (V9 region of the SSU rRNA gene) communities in 21 activated sludge samples obtained from full-scale municipal WWTPs in Germany. Microbial community patterns were compared to standard WWTP operating parameters and two traditionally used WWTP performance indicators (Sludge Biotic Index and Sludge Index). Both indices showed low concordance and hardly correlated with chemical WWTP performance parameters nor did they correlate with microbial community structures. In contrast, microbial community profiles significantly correlated with WWTP performance parameters and operating conditions of the plants under study. Therefore, eDNA metabarcode profiles of whole microbial communities indicate the performance of WWTP and can provide useful information for management strategies. We here suggest a strategy for the development of an eDNA metabarcode based bioindicator system, which can be implemented in future standard monitoring programs for WWTP performance and effluent quality.