Structural insights into coordinating 5S RNP rotation with ITS2 pre-RNA processing during ribosome formation.

The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation. Upon rixosome positioning, the other sub-module with Las1 endonuclease and Grc3 polynucleotide-kinase can reach a strategic position at the pre-60S foot to cleave and 5' phosphorylate the nearby ITS2 pre-rRNA. Finally, inward movement of the L1 stalk permits the flexible Nop53 N-terminus with its AIM motif to become positioned at the base of the L1-stalk to facilitate Mtr4 helicase-exosome participation for completing ITS2 removal. Thus, the rixosome structure elucidates the coordination of two central ribosome biogenesis events, but its role in gene silencing may adapt similar strategies.

Symbiodiniaceae diversity varies by host and environment across thermally distinct reefs.

Endosymbiotic dinoflagellates (Symbiodiniaceae) influence coral thermal tolerance at both local and regional scales. In isolation, the effects of host genetics, environment, and thermal disturbances on symbiont communities are well understood, yet their combined effects remain poorly resolved. Here, we investigate Symbiodiniaceae across 1300 km in Australia's Coral Sea Marine Park to disentangle these interactive effects. We identified Symbiodiniaceae to species-level resolution for three coral species (Acropora cf humilis, Pocillopora verrucosa, and Pocillopora meandrina) by sequencing two genetic markers of the symbiont (ITS2 and psbAncr), paired with genotype-by-sequencing of the coral host (DArT-seq). Our samples predominantly returned sequences from the genus Cladocopium, where Acropora cf humilis affiliated with C3k, Pocillopora verrucosa with C. pacificum, and Pocillopora meandrina with C. latusorum. Multivariate analyses revealed that Acropora symbionts were driven strongly by local environment and thermal disturbances. In contrast, Pocillopora symbiont communities were both partitioned 2.5-fold more by host genetic structure than by environmental structure. Among the two Pocillopora species, the effects of environment and host genetics explained four times more variation in symbionts for P. meandrina than P. verrucosa. The concurrent bleaching event in 2020 had variable impacts on symbiont communities, consistent with patterns in P. verrucosa and A. cf humilis, but not P. meandrina. Our findings demonstrate how symbiont macroscale community structure responses to environmental gradients depend on host species and their respective population structure. Integrating host, symbiont, and environmental data will help forecast the adaptive potential of corals and their symbionts amidst a rapidly changing environment.

Cytomolecular trends in Chamaecrista Moench (Caesalpinioideae, Leguminosae) diversification.

Chamaecrista is a Pantropical legume genus of the tribe Cassieae, which includes six other genera. In contrast to most of the other Cassieae genera, Chamaecrista shows significant variability in chromosome number (from 2n = 14 to 2n = 56), with small and morphologically similar chromosomes. Here, we performed a new cytomolecular analysis on chromosome number, genome size, and rDNA site distribution in a molecular phylogenetic perspective to interpret the karyotype trends of Chamaecrista and other two genera of Cassieae, seeking to understand their systematics and evolution. Our phylogenetic analysis revealed that Chamaecrista is monophyletic and can be divided into four major clades corresponding to the four sections of the genus. Chromosome numbers ranged from 2n = 14, 16 (section Chamaecrista) to 2n = 28 (sections Absus, Apoucouita, and Baseophyllum). The number of 5S and 35S rDNA sites varied between one and three pairs per karyotype, distributed on different chromosomes or in synteny, with no obvious phylogenetic significance. Our data allowed us to propose x = 7 as the basic chromosome number of Cassieae, which was changed by polyploidy generating x = 14 (sections Absus, Apoucouita, and Baseophyllum) and by ascending dysploidy to x = 8 (section Chamaecrista). The DNA content values supported this hypothesis, with the genomes of the putative tetraploids being larger than those of the putative diploids. We hypothesized that ascending dysploidy, polyploidy, and rDNA amplification/deamplification are the major events in the karyotypic diversification of Chamaecrista. The chromosomal marks characterized here may have cytotaxonomic potential in future studies.

Population genetic structure and phylogenetic analysis of Anopheles hyrcanus (Diptera: Culicidae) inferred from DNA sequences of nuclear ITS2 and the mitochondrial COI gene in the northern part of Iran.

BACKGROUND: The Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide. METHODS: Mosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software. RESULTS: In this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity. CONCLUSIONS: An. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon.

Molecular authentication of commercial "Qian-hu" through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature.

BACKGROUND: Peucedani Radix, also known as "Qian-hu" is a traditional Chinese medicine derived from Peucedanum praeruptorum Dunn. It is widely utilized for treating wind-heat colds and coughs accompanied by excessive phlegm. However, due to morphological similarities, limited resources, and heightened market demand, numerous substitutes and adulterants of Peucedani Radix have emerged within the herbal medicine market. Moreover, Peucedani Radix is typically dried and sliced for sale, rendering traditional identification methods challenging. MATERIALS AND METHODS: We initially examined and compared 104 commercial "Qian-hu" samples from various Chinese medicinal markets and 44 species representing genuine, adulterants or substitutes, utilizing the mini barcode ITS2 region to elucidate the botanical origins of the commercial "Qian-hu". The nucleotide signature specific to Peucedani Radix was subsequently developed by analyzing the polymorphic sites within the aligned ITS2 sequences. RESULTS: The results demonstrated a success rate of 100% and 93.3% for DNA extraction and PCR amplification, respectively. Forty-five samples were authentic "Qian-hu", while the remaining samples were all adulterants, originating from nine distinct species. Peucedani Radix, its substitutes, and adulterants were successfully identified based on the neighbor-joining tree. The 24-bp nucleotide signature (5'-ATTGTCGTACGAATCCTCGTCGTC-3') revealed distinct differences between Peucedani Radix and its common substitutes and adulterants. The newly designed specific primers (PR-F/PR-R) can amplify the nucleotide signature region from commercial samples and processed materials with severe DNA degradation. CONCLUSIONS: We advocate for the utilization of ITS2 and nucleotide signature for the rapid and precise identification of herbal medicines and their adulterants to regulate the Chinese herbal medicine industry.

Identification and distribution of Rhipicephalus microplus in selected high-cattle density districts in Uganda: signaling future demand for novel tick control approaches.

BACKGROUND: Rhipicephalus (Boophilus) microplus (Canestrini, 1888), the Asian blue tick, is a highly invasive and adaptable ectoparasite. This tick species has successfully established itself in most regions of the world, with movement of cattle being a major driver for its spread. In the recent past, R. microplus ticks have been reported in three districts of Uganda. Information on its spread and distribution are vital in deepening our understanding of the ecological scenarios that lead to tick persistence and in the formulation of control strategies. This is especially important in the cattle-dense districts. METHODS: We randomly collected tick specimens from 1,461cattle spread across seven cattle dense districts located in the Central, Karamoja and West Nile regions of Uganda from January to September 2020. The ticks were identified using standard morpho-taxonomic keys and the R. microplus tick species identities were confirmed by sequencing of the ITS2 region, 12S rRNA and 16S rRNA genes and phylogenetic analyses. RESULTS: Adult ticks (n = 13,019) were collected from 1,461 cattle. Seventeen tick species were identified based on morpho-taxonomic keys and the majority (47.4%; n=6184) of these were R. appendiculatus. In total, 257 R. microplus ticks were found infesting cattle in 18 study sites in the districts of Amudat, Kaabong, Napak (Karamoja region) and Arua (West Nile region). The identity of R. microplus was confirmed using molecular technics. No R. microplus tick was recorded in the districts of Lyantonde and Nakaseke (Central region). Arua district accounted for 82.1% (n=211) of the R. microplus ticks recorded followed by Napak district at 16.3% (n=42), while Amudat and Kaabong districts accounted for 1.5% (n=4). Rhipicephalus microplus and R. decoloratus co-existed in 6 of the 13 study sites in Arua district, while in another 6 study sites, no R. decoloratus was recorded. In the Karamoja region districts R. decoloratus co-existed with R.microplus. Of the total 618 ticks belonging to four species of the subgenus Boophilus recorded in this study, R. decoloratus accounted for 50.04% (n=334), followed by R. microplus at 41.58% (n=257), R. geigyi at 2.75% (n=17) and R. annulatus at 1.61% (n=10). In the districts of Amudat, Kaabong and Napak, R. decoloratus was more dominant (76.1%; n=179) of the three Rhipicephalus (Boophilus) tick species recorded, followed by R. microplus (19.5%; n=46) and R. geigyi (4.2%; n=10). Contrariwise, R. microplus was more dominant (84%; n=211) in Arua district followed by R. decoloratus (10.7%; n=27), R. annulatus (3.9%; n=10) and R. geigyi (1.1%; n=3). Phylogenetic analyses of the ITS2 region, 12S rRNA and 16S rRNA genes revealed subgrouping of the obtained sequences with the previously published R. microplus sequences from other parts of the world. CONCLUSION: Rhipicephalus microplus ticks were found infesting cattle in four districts of Uganda. The inability to find R. decoloratus, an indigenous tick, from six sites in the district of Arua is suggestive of its replacement by R. microplus. Rhipicephalus microplus negatively affects livestock production, and therefore, there is a need to determine its distribution and to deepen the understanding of the ecological factors that lead to its spread and persistence in an area.

First record of Anopheles claviger (Meigen, 1804) (Diptera, Culicidae) in Karelia, Northwestern Russia.

Anopheles claviger (Meigen, 1804) (Diptera, Culicidae) is widespread in the western Palaearctic Region, but it was recorded in Karelia (Russia) for the first time. This record is one of the northernmost ones in the Palaearctic Region and Russia, updates the northern border of the An. claviger range. Mosquitoes were collected from July to September 2023 in the southern Karelia (the village of Gomselga, Kondopoga District, and Petrozavodsk) using Krishtal trap (from human) and Mosquito Magnet® trap (Pioneer design, Octenol as attractant). Seven females of An. claviger were collected in Gomselga; one specimen was sampled from Petrozavodsk City parks. Morphological identification of eight females was verified by COI and ITS2 sequences. Phylogenetic analysis of ITS2 and COI sequences confirmed the collected specimens to An. claviger s. s., clustering in both cases in a strongly supported clade clearly differentiated from the closely related species An. petragnani. The high diversity of An. claviger haplotypes from Karelia is in agreement with data from other geographical regions and shows that the records of this species in Gomselga and Petrozavodsk are not accidental.

Survey of Phytophthora Diversity Reveals P. abietivora as a Potential Phytophthora Root Rot Pathogen in Québec Christmas Tree Plantations.

Christmas trees are an economically and culturally important ornamental plant in North America. Many microorganisms are pathogens of firs cultivated as Christmas trees. Among those, Phytophthora causes millions of dollars in damage to plantations annually. In Canada, it is unknown which species are responsible for Phytophthora root rot (PRR) of cultivated Abies species. Between 2019 and 2021, soil and root samples were collected from 40 Christmas tree plantations in Québec province. We used soil baiting and direct isolation from unidentified root fragments to assess the diversity of culturable Phytophthora spp. The obtained isolates were identified using a multilocus sequencing and phylogenetic approach. A total of 44 isolates were identified, including eight P. chlamydospora, eight P. abietivora, seven P. gonapodyides, three P. gregata, six P. megasperma, and two P. kelmanii isolates, plus 10 isolates belonging to a previously unknown taxon that is phylogenetically close to P. chlamydospora and P. gonapodyides. Among the known species, P. abietivora was the most prevalent isolated species associated with trees showing aboveground PRR-like symptoms. Pathogenicity trials confirmed the pathogenicity potential of P. abietivora on both Fraser fir and balsam fir seedlings. Our study provides a first snapshot of the Phytophthora diversity in Québec's Christmas tree productions and describes multiple potential first associations between Phytophthora species and Abies balsamea and A. fraseri.[Formula: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Natural Resources Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Integrative taxonomy of Anaporrhutum mundae sp. nov. (Trematoda: Gorgoderidae), a parasite of the Munda round ray Urotrygon munda (Urotrygonidae) in Costa Rica.

A new species of trematode of anaporrhutine gorgoderid, from the gill chambers of the Munda round ray Urotrygon munda in Costa Rica is described, based on an integrative taxonomic approach that includes the use of light and scanning electron microscopy, ITS2 and 28S rDNA sequencing, and phylogenetic analysis. Anaporrhutum mundae sp. nov. can be distinguished from congeneric species by a combination of morphological traits and particularly by having the genital pore opening at the level of the intestinal bifurcation. The new species also can be distinguished from all other species of Anaporrhutum, except A. euzeti Curran, Blend & Overstreet, 2003, by having fewer testicular follicles per testis. Anaporrhutum mundae sp. nov. also differs from A. euzeti in its forebody shape and by having different morphology and location of the vitellaria. The study of the tegumental surface of A. mundae sp. nov., as revealed by scanning electron microscopy, allowed detection of new morphological characters for a member of Anaporrhutinae that may be of taxonomic value. These are: a stylet cavity dorsal to the oral sucker with a large penetration gland opening on each side of the cavity and small penetration gland openings located ventral to the stylet cavity, arranged in a circle around the mouth. This represents the first record of an Anaporrhutum species from Costa Rica. Further, A. mundae sp. nov. represents the first parasite described or reported in this host.

Morphological, ultrastructural, and phylogenetic analysis of Ascaridia columbae infecting domestic pigeons (Columba livia domestica).

Ascaridia species are the most common nematodes infecting pigeons. The current study investigated specific identity of nematode parasites collected from domestic pigeons (Columba livia domestica) in Al-Qassim Region, Saudi Arabia. Out of 354 pigeons, 13.3 % were infected with nematode parasites. The morphological structure and genetic relationship of nematode worms were studied using conventional methods (Light and scanning electron microscopes) coupled with the newly introduced molecular method. Microscopical and ultrastructure observations showed that the present nematode worms belong to the genus Ascaridia and have all the characteristic features of Ascaridia columbae. Moreover, Random Amplifier morphometric (RAPD) PCR analysis revealed that the present A. columbae had a close identity of up to 98.3 % to Ascaridia columbae JX624729 for Cox-1 gene regions, and up to 98.3 % to Ascaridia nymphii LC057210, and Ascaridia galli EF180058 for ITS1-5.8s- ITS2 rDNA gene regions. Phylogenetic analysis supported the placement of this Ascaridia species within Ascaridiidae family with close relationships to other nematode species obtained from GenBank. Finally, our study recommends using molecular analysis in helminths identification as the main methodology for correct identification especially in closely related species.

Molecular Tools for Early Detection of Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Reports of the expansion of the Asia malaria vector Anopheles stephensi mosquito into new geographic areas are increasing, which poses a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographic areas is key to containing and controlling this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi mosquitoes, we developed a species-specific PCR and a real-time PCR targeting a unique segment of the second internal transcribed spacer lacking homology to any other organism. Both PCRs can be used to identify An. stephensi mosquitoes individually or in pooled samples of mixed species, including when present in extremely low proportions (1:500). This study also reports a method for selective amplification and sequencing of partial ribosomal DNA from An. stephensi mosquitoes for their confirmation in pooled samples of mixed species.

Ocular Trematodiasis in Children, Sri Lanka.

Using histopathology and phylogenetic analysis of the internal transcribed spacer 2 gene, we found >2 distinct trematode species that caused ocular trematode infections in children in Sri Lanka. Collaborations between clinicians and parasitologists and community awareness of water-related contamination hazards will promote diagnosis, control, and prevention of ocular trematode infections.

GC heterogeneity reveals sequence-structures evolution of angiosperm ITS2.

BACKGROUND: Despite GC variation constitutes a fundamental element of genome and species diversity, the precise mechanisms driving it remain unclear. The abundant sequence data available for the ITS2, a commonly employed phylogenetic marker in plants, offers an exceptional resource for exploring the GC variation across angiosperms. RESULTS: A comprehensive selection of 8666 species, comprising 165 genera, 63 families, and 30 orders were used for the analyses. The alignment of ITS2 sequence-structures and partitioning of secondary structures into paired and unpaired regions were performed using 4SALE. Substitution rates and frequencies among GC base-pairs in the paired regions of ITS2 were calculated using RNA-specific models in the PHASE package. The results showed that the distribution of ITS2 GC contents on the angiosperm phylogeny was heterogeneous, but their increase was generally associated with ITS2 sequence homogenization, thereby supporting the occurrence of GC-biased gene conversion (gBGC) during the concerted evolution of ITS2. Additionally, the GC content in the paired regions of the ITS2 secondary structure was significantly higher than that of the unpaired regions, indicating the selection of GC for thermodynamic stability. Furthermore, the RNA substitution models demonstrated that base-pair transformations favored both the elevation and fixation of GC in the paired regions, providing further support for gBGC. CONCLUSIONS: Our findings highlight the significance of secondary structure in GC investigation, which demonstrate that both gBGC and structure-based selection are influential factors driving angiosperm ITS2 GC content.

Characterization of the intestinal fungal microbiome in patients with hepatocellular carcinoma.

OBJECTIVE: Gut mycobiota plays a crucial role in benign liver diseases; however, its correlation with hepatocellular carcinoma (HCC) remains elusive. This study aimed to elucidate fungal differences in patients with HCC-associated cirrhosis compared to cirrhotic patients without HCC and healthy controls. METHODS: The 72 fecal samples from 34 HCC patients, 20 cirrhotic patients, and 18 healthy controls were collected and analyzed using ITS2 rDNA sequencing. RESULTS: Our results revealed the presence of intestinal fungal dysbiosis with significant enrichment of opportunistic pathogenic fungi such as Malassezia, Malassezia sp., Candida, and C. albicans in HCC patients compared with healthy controls and cirrhosis patients. Alpha-diversity analysis demonstrated that patients with HCC and cirrhosis showed decreased fungal diversity compared to healthy controls. Beta diversity analysis indicated that the three groups exhibited significant segregated clustering. Besides, C. albicans was found to be significantly more abundant in the HCC patients with TNM stage III-IV than those with stage I-II, in contrast to the commensal organism S. cerevisiae. We also confirmed that the HCC patients were successfully classified with an area under the curve value of 0.906 based on the fecal fungal signature. Finally, our animal experiments confirm that aberrant colonization of the intestine by C. albicans and M. furfur can promote the development of HCC. CONCLUSIONS: This study indicates that dysbiosis of the gut mycobiome might be involved in HCC development. TRIAL REGISTRATION: ChiCTR, ChiCTR2100054537. Registered 19 December 2021, http://www.chictr.org.cn/edit.aspx?pid=144550&htm=4.

Root-associated fungal communities are influenced more by soils than by plant-host root traits in a Chinese tropical forest.

Forest fungal communities are shaped by the interactions between host tree root systems and the associated soil conditions. We investigated how the soil environment, root morphological traits, and root chemistry influence root-inhabiting fungal communities in three tropical forest sites of varying successional status in Xishuangbanna, China. For 150 trees of 66 species, we measured root morphology and tissue chemistry. Tree species identity was confirmed by sequencing rbcL, and root-associated fungal (RAF) communities were determined using high-throughput ITS2 sequencing. Using distance-based redundancy analysis and hierarchical variation partitioning, we quantified the relative importance of two soil variables (site average total phosphorus and available phosphorus), four root traits (dry matter content, tissue density, specific tip abundance, and forks), and three root tissue elemental concentrations (nitrogen, calcium, and manganese) on RAF community dissimilarity. The root and soil environment collectively explained 23% of RAF compositional variation. Soil phosphorus explained 76% of that variation. Twenty fungal taxa differentiated RAF communities among the three sites. Soil phosphorus most strongly affects RAF assemblages in this tropical forest. Variation in root calcium and manganese concentrations and root morphology among tree hosts, principally an architectural trade-off between dense, highly branched vs less-dense, herringbone-type root systems, are important secondary determinants.

Pollen DNA metabarcoding reveals cryptic diversity and high spatial turnover in alpine plant-pollinator networks.

Alpine plant-pollinator communities play an important role in the functioning of alpine ecosystems, which are highly threatened by climate change. However, we still have a poor understanding of how environmental factors and spatiotemporal variability shape these communities. Here, we investigate what drives structure and beta diversity in a plant-pollinator metacommunity from the Australian alpine region using two approaches: pollen DNA metabarcoding (MB) and observations. Individual pollinators often carry pollen from multiple plant species, and therefore we expected MB to reveal a more diverse and complex network structure. We used two gene regions (ITS2 and trnL) to identify plant species present in the pollen loads of 154 insect pollinator specimens from three alpine habitats and construct MB networks, and compared them to networks based on observations alone. We compared species and interaction turnover across space for both types of networks, and evaluated their differences for plant phylogenetic diversity and beta diversity. We found significant structural differences between the two types of networks; notably, MB networks were much less specialized but more diverse than observation networks, with MB detecting many cryptic plant species. Both approaches revealed that alpine pollination networks are very generalized, but we estimated a high spatial turnover of plant species (0.79) and interaction rewiring (0.6) as well as high plant phylogenetic diversity (0.68) driven by habitat differences based on the larger diversity of plant species and species interactions detected with MB. Overall, our findings show that habitat and microclimatic heterogeneity drives diversity and fine-scale spatial turnover of alpine plant-pollinator networks.

New evidence of consistency between phylogeny and morphology for two taxa in ciliated protists, the subclasses Oligotrichia and Choreotrichia (Protista, Ciliophora).

Marine planktonic ciliates are largely oligotrichs and choreotrichs, which are two subclasses of the class Spirotrichea. The current phylogenetic assignments of oligotrichs and choreotrichs are inconsistent with previous results based on morphological features, probably hindered by the limited information from a single gene locus. Here we provide 53 new sequences from small subunit ribosomal RNA (SSU rDNA), ITS1-5.8S rDNA-ITS2, and large subunit ribosomal RNA (LSU rDNA) gene loci in 25 oligotrich and choreotrich species. We also predict RNA secondary structures for the ITS2 regions in 55 species, 48 species of which are reported for the first time. Based on these novel data, we make a more comprehensive phylogenetic reconstruction, revealing consistency between morphological taxonomy and an updated phylogenetic system for oligotrichs and choreotrichs. With the addition of data from ciliature patterns and genes, the phylogenetic analysis of the subclass Oligotrichia suggests three evolutionary trajectories, among which: 1) Novistrombidium asserts an ancestral ciliary pattern in Oligotrichia; 2) the subgenera division of Novistrombidium and Parallelostrombidium are fully supported; 3) the three families (Tontoniidae, Pelagostrombidiidae and Cyrtostrombidiidae) all evolved from the most diverse family Strombidiidae, which explains why strombidiids consistently form polyphyletic clades. In the subclass Choreotrichia, Strombidinopsis likely possesses an ancestral position to other choreotrichs, and both phylogenetic analysis and RNA secondary structure prediction support the hypothesis that tintinnids may have evolved from Strombidinopsis. The results presented here offer an updated hypothesis for the evolutionary history of oligotrichs and choreotrichs based on new evidence obtained by expanding sampling of molecular information across multiple gene loci.

Paramecium: RNA sequence-structure phylogenetics.

Organisms classified as members of the genus Paramecium belong to the best-known group of single-celled eukaryotes. Nevertheless, the phylogeny within the genus Paramecium has been discussed and revisited in recent decades and remains partly unresolved. By applying an RNA sequence-structure approach, we attempt to increase accuracy and robustness of phylogenetic trees. For each individual 18S and internal transcribed spacer 2 (ITS2) sequence, a putative secondary structure was predicted through homology modelling. While searching for a structural template, we found, in contrast to the available literature, that the ITS2 molecule consists of three helices in members of the genus Paramecium and four helices in members of the genus Tetrahymena. Two sequencestructure neighbor-joining overall trees were reconstructed with (1) more than 400 taxa (ITS2) and (2) more than 200 taxa (18S). For smaller subsets, neighbor-joining, maximum-parsimony, and maximum-likelihood analyses were executed using sequence-structure information simultaneously. Based on a combined data set (ITS2+18S rDNA) a well-supported tree was reconstructed with bootstrap values over 50 in at least one of the applied analyses. Our results are in general agreement with those published in the available literature based on multi-gene analyses. Our study supports the simultaneous use of sequence-structure data to reconstruct accurate and robust phylogenetic trees.

Verminotic pneumonia in South American fur seal (Arctocephalus australis) in Southern Brazil.

Verminotic pneumonia caused by Parafilaroides spp. nematodes is an underreported disease in beached South American fur seals, with scant literature available on the characteristics of parafilaroidiasis, the nematode itself, as well as its occurrence in pinnipeds in Brazil. The present work aims to identify, describe and detail the histological features of the infection and molecular characteristics of verminotic pneumonia in the South American fur seal. Twenty-six specimens of Arctocephalus australis, found dead on the northern coast of Rio Grande do Sul in 2021, were analysed. These animals were identified and submitted to necropsy and histology. For the molecular identification of metastrongylids, lung fragments were subjected to DNA extraction, polymerase chain reaction targeting the Internal transcribed spacer 2 (ITS-2) gene and subsequent sequencing. In total, 12 animals presented with parasites in the lung parenchyma on histological evaluation, and only 1 showed a granulomatous lung lesion at necropsy. Microscopically, the nematodes were found mainly in the alveoli, associated with little or no inflammatory response, and they had morphological characteristics compatible with metastrongylids. Six ITS-2 gene quality sequences were obtained; after comparative analysis via BLAST, they showed similarity with sequences obtained from Parafilaroides sp. Therefore, verminotic pneumonia caused by Parafilaroides represents an important differential diagnosis of lung disease in South American fur seals found on the northern coast of Rio Grande do Sul.

Refugia or reservoir? Feral goats and their role in the maintenance and circulation of benzimidazole-resistant gastrointestinal nematodes on shared pastures.

Gastrointestinal nematodes threaten the productivity of grazing livestock and anthelmintic resistance has emerged globally. It is broadly understood that wild ruminants living in sympatry with livestock act as a positive source of refugia for anthelmintic-susceptible nematodes. However, they might also act as reservoirs of anthelmintic-resistant nematodes, contributing to the spread of anthelmintic resistance at a regional scale. Here, we sampled managed sheep and cattle together with feral goats within the same property in New South Wales, Australia. Internal transcribed spacer 2 (ITS-2) nemabiome metabarcoding identified 12 gastrointestinal nematodes (Cooperia oncophora, Cooperia punctata, Haemonchus contortus, Haemonchus placei, Nematodirus spathiger, Ostertagia ostertagi, Teladorsagia circumcincta, Oesophagostomum radiatum, Oesophagostomum venulosum, Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus rugatus). Isotype-1 ß-tubulin metabarcoding targeting benzimidazole resistance polymorphisms identified 6 of these nematode species (C. oncophora, C. punctata, H. contortus, H. placei, O. ostertagi and T. circumcincta), with the remaining 3 genera unable to be identified to the species level (Nematodirus, Oesophagostomum, Trichostrongylus). Both ITS-2 and ß-tubulin metabarcoding showed the presence of a cryptic species of T. circumcincta, known from domestic goats in France. Of the gastrointestinal nematodes detected via ß-tubulin metabarcoding, H. contortus, T. circumcincta, Nematodirus and Trichostrongylus exhibited the presence of at least one resistance genotype. We found that generalist gastrointestinal nematodes in untreated feral goats had a similarly high frequency of the benzimidazole-resistant F200Y polymorphism as those nematodes in sheep and cattle. This suggests cross-transmission and maintenance of the resistant genotype within the wild ruminant population, affirming that wild ruminants should be considered potential reservoirs of anthelmintic resistance.