A fungi hotspot deep in the ocean: explaining the presence of Gjaerumia minor in equatorial Pacific bathypelagic waters.

A plant parasite associated with the white haze disease in apples, the Basidiomycota Gjaerumia minor, has been found in most samples of the global bathypelagic ocean. An analysis of environmental 18S rDNA sequences on 12 vertical profiles of the Malaspina 2010 expedition shows that the relative abundance of this cultured species increases with depth while its distribution is remarkably different between the deep waters of the Pacific and Atlantic oceans, being present in higher concentrations in the former. This is evident from sequence analysis and a microscopic survey with a species-specific newly designed TSA-FISH probe. Several hints point to the hypothesis that G. minor is transported to the deep ocean attached to particles, and the absence of G. minor in bathypelagic Atlantic waters could then be explained by the absence of this organism in surface waters of the equatorial Atlantic. The good correlation of G. minor biomass with Apparent Oxygen Utilization, recalcitrant carbon and free-living prokaryotic biomass in South Pacific waters, together with the identification of the observed cells as yeasts and not as resting spores (teliospores), point to the possibility that once arrived at deep layers this species keeps on growing and thriving.

Myxobolus acanthogobii Hoshina, 1952 and Myxobolus selari n. sp. (Myxosporea: Myxobolidae) infecting brain of commercial fishes in Terengganu, Malaysia.

Myxosporean infection in marine water fishes has drawn less attention than in freshwater fishes, which resulted in a higher taxonomic variety in freshwater in Malaysia. This study aimed to address the gap by conducting a myxosporean survey on two commercially significant marine fish species, Nemipterus furcosus (Valenciennes) (Eupercaria incertae sedis: Nemipteridae) and Selar crumenophthalmus (Bloch) (Carangiformes: Carangidae), collected from the northeastern part of peninsular Malaysia. During the examination of the organs, two distinct Myxobolus Bütschli, 1882 species were discovered in the brain tissue of these fishes, despite the absence of any observable pathological signs. The two Myxobolus species were characterized through morphometry, morphology, and analysis of partial small subunit ribosomal RNA (18S rDNA) gene. As a result, Myxobolus acanthogobii Hoshina, 1952, which infects 2.3% of N. furcosus, is synonymous with a myxobolid species commonly found in Japanese waters, based on its morphological traits, tissue tropism, and molecular diagnostics. Furthermore, a novel species, Myxobolus selari n. sp., was described, infecting the brain of one (11%) individual S. crumenophthalmus. This unique species displayed distinctive features, placing it within a well-supported subclade primarily comprising brain-infecting myxobolids. Maximum likelihood analysis further revealed the close relationships among these brain-infecting myxobolids, underscoring the significance of tissue tropism and host taxonomy for myxobolids. This study represents the initial documentation of Myxobolus species within the southern South China Sea, shedding light on the potential diversity of marine myxosporean in this region. This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:7C400E35-7CB8-4DEE-92B7-F75FF3926441.

Morphological and molecular characteristics of a Trypanosoma sp. from triatomines (Triatoma rubrofasciata) in China.

BACKGROUND: Triatomines (kissing bugs) are natural vectors of trypanosomes, which are single-celled parasitic protozoans, such as Trypanosoma cruzi, T. conorhini and T. rangeli. The understanding of the transmission cycle of T. conorhini and Triatoma rubrofasciata in China is not fully known. METHODS: The parasites in the faeces and intestinal contents of the Tr. rubrofasciata were collected, and morphology indices were measured under a microscope to determine the species. DNA was extracted from the samples, and fragments of 18S rRNA, heat shock protein 70 (HSP70) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) were amplified and sequenced. The obtained sequences were then identified using the BLAST search engine, followed by several phylogenetic analyses. Finally, laboratory infections were conducted to test whether Tr. rubrofasciata transmit the parasite to rats (or mice) through bites. Moreover, 135 Tr. rubrofasciata samples were collected from the Guangxi region and were used in assays to investigate the prevalence of trypanosome infection. RESULTS: Trypanosoma sp. were found in the faeces and intestinal contents of Tr. rubrofasciata, which were collected in the Guangxi region of southern China and mostly exhibited characteristics typical of epimastigotes, such as the presence of a nucleus, a free flagellum and a kinetoplast. The body length ranged from 6.3 to 33.9 µm, the flagellum length ranged from 8.7 to 29.8 µm, the nucleus index was 0.6 and the kinetoplast length was -4.6. BLAST analysis revealed that the 18S rRNA, HSP70 and gGAPDH sequences of Trypanosoma sp. exhibited the highest degree of similarity with those of T. conorhini (99.7%, 99.0% and 99.0%, respectively) and formed a well-supported clade close to T. conorhini and T. vespertilionis but were distinct from those of T. rangeli and T. cruzi. Laboratory experiments revealed that both rats and mice developed low parasitaemia after inoculation with Trypanosoma sp. and laboratory-fed Tr. rubrofasciata became infected after feeding on trypanosome-positive rats and mice. However, the infected Tr. rubrofasciata did not transmit Trypanosoma sp. to their offspring. Moreover, our investigation revealed a high prevalence of Trypanosoma sp. infection in Tr. rubrofasciata, with up to 36.3% of specimens tested in the field being infected. CONCLUSIONS: Our study is the first to provide a solid record of T. conorhini from Tr. rubrofasciata in China with morphological and molecular evidence. This Chinese T. conorhini is unlikely to have spread through transovarial transmission in Tr. rubrofasciata, but instead, it is more likely that the parasite is transmitted between Tr. rubrofasciata and mice (or rats). However, there was a high prevalence of T. conorhini in the Tr. rubrofasciata from our collection sites and numerous human cases of Tr. rubrofasciata bites were recorded. Moreover, whether these T. conorhini strains are pathogenic to humans has not been investigated.

The effect of the 13C abundance of soil microbial DNA on identifying labelled fractions after ultracentrifugation.

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ13C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ13C values while adding only 5 atom% 13C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ13C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP. KEY POINTS: • Appropriate 13C-DNA amount was needed for DNA-SIP. • Detecting the 13C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions. • Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled.

Succession of diversity, assembly mechanisms, and activities of the microeukaryotic community throughout Scrippsiella acuminata (Dinophyceae) bloom phases.

Harmful algal bloom (HAB) is a rapidly expanding marine ecological hazard. Although numerous studies have been carried out about the ecological impact and the ecological mechanism of HAB outbreaks, few studies have comprehensively addressed the shifts of species composition, metabolic activity level, driving factors and community assembly mechanisms of microeukaryotic plankton in the course of the bloom event. To fill the gap of research, we conducted 18S ribosomal DNA and RNA sequencing during the initiation, development, sustenance and decline stages of a Scrippsiella acuminata (S. acuminata) bloom at the coastal sea of Fujian Province, China. We found that the bloom event caused a decrease in microeukaryotic plankton species diversity and increase in community homogeneity. Our results revealed that the RNA- and DNA-inferred communities were similar, but α-diversity was more dynamic in RNA- than in DNA-inferred communities. The main taxa with high projected metabolic activity (with RNA:DNA ratio as the proxy) during the bloom included dinoflagellates, Cercozoa, Chlorophyta, Protalveolata, and diatoms. The role of deterministic processes in microeukaryotic plankton community assembly increased during the bloom, but stochastic processes were always the dominant assembly mechanism throughout the bloom process. Our findings improve the understanding of temporal patterns, driving factors and assembly mechanisms underlying the microeukarytic plankton community in a dinoflagellate bloom.

Persistent and multiclonal malaria parasite dynamics despite extended artemether-lumefantrine treatment in children.

Standard diagnostics used in longitudinal antimalarial studies are unable to characterize the complexity of submicroscopic parasite dynamics, particularly in high transmission settings. We use molecular markers and amplicon sequencing to characterize post-treatment stage-specific malaria parasite dynamics during a 42 day randomized trial of 3- versus 5 day artemether-lumefantrine in 303 children with and without HIV (ClinicalTrials.gov number NCT03453840). The prevalence of parasite-derived 18S rRNA is >70% in children throughout follow-up, and the ring-stage marker SBP1 is detectable in over 15% of children on day 14 despite effective treatment. We find that the extended regimen significantly lowers the risk of recurrent ring-stage parasitemia compared to the standard 3 day regimen, and that higher day 7 lumefantrine concentrations decrease the probability of ring-stage parasites in the early post-treatment period. Longitudinal amplicon sequencing reveals remarkably dynamic patterns of multiclonal infections that include new and persistent clones in both the early post-treatment and later time periods. Our data indicate that post-treatment parasite dynamics are highly complex despite efficacious therapy, findings that will inform strategies to optimize regimens in the face of emerging partial artemisinin resistance in Africa.

18S rDNA sequence-structure phylogeny of the eukaryotes simultaneously inferred from sequences and their individual secondary structures.

OBJECTIVE: The eukaryotic tree of life has been subject of numerous studies ever since the nineteenth century, with more supergroups and their sister relations being decoded in the last years. In this study, we reconstructed the phylogeny of eukaryotes using complete 18S rDNA sequences and their individual secondary structures simultaneously. After the sequence-structure data was encoded, it was automatically aligned and analyzed using sequence-only as well as sequence-structure approaches. We present overall neighbor-joining trees of 211 eukaryotes as well as the respective profile neighbor-joining trees, which helped to resolve the basal branching pattern. A manually chosen subset was further inspected using neighbor-joining, maximum parsimony, and maximum likelihood analyses. Additionally, the 75 and 100 percent consensus structures of the subset were predicted. RESULTS: All sequence-structure approaches show improvements compared to the respective sequence-only approaches: the average bootstrap support per node of the sequence-structure profile neighbor-joining analyses with 90.3, was higher than the average bootstrap support of the sequence-only profile neighbor-joining analysis with 73.9. Also, the subset analyses using sequence-structure data were better supported. Furthermore, more subgroups of the supergroups were recovered as monophyletic and sister group relations were much more comparable to results as obtained by multi-marker analyses.

Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools. METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity. RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested. CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

Sample design in biodiversity studies matters: a fine-scale study of Lawrence's velvet worm, Peripatopsis lawrencei (Onychophora: Peripatopsidae), reveals hidden diversity.

A fine-scale phylogenetic and phylogeographic analysis of Peripatopsis lawrencei s.l. was conducted with both mitochondrial and nuclear DNA sequence data, using both external morphology and scanning electron microscopy of taxonomically important characters. A total of 119 sequences were used for the mitochondrial cytochrome c oxidase subunit I (COI ) whereas a single representative specimen from each locality was sequenced for the nuclear 18S rRNA locus. Phylogenetic analyses were conducted on the total COI data set and the combined COI + 18S rRNA data set using a Bayesian analysis and maximum likelihood analyses. For the combined DNA sequence data set, a divergence time estimation was further undertaken in BEAST and specimens placed in a phylogenetic framework including all the described Peripatopsis species from South Africa. In addition, a phylogeographic study was conducted exclusively on P. lawrencei s.s. (clade A) using an analysis of molecular variance and haplotype network. Phylogenetic results indicated that, at the Oubos sample locality, two highly distinct genetic lineages were present (clades A and B), whereas a divergence time estimation suggests a Miocene cladogenesis of the novel Oubos lineage. Marked phylogeographic structure was observed for P. lawrencei s.s. (restricted to clade A) across the distribution range with limited maternal dispersal. Morphologically, the two sympatric lineages at Oubos A and B differed in leg pair number, ventral colour and dorsal scale rank counts, as evident from scanning electron microscopy. Our results support the recognition of a distinct species that occurs in sympatry with P. lawrencei s.s. The new species, P. aereus sp. nov. (clade B) is described and the implication for fine-scale taxonomic studies on saproxylic taxa is discussed. ZooBank: urn:lsid:zoobank.org:pub:AB6E0BDA-7B5F-4FD3-A863-BA7C814E278C.

Morphological and molecular characterization of Eimeria haematopusi n. sp. (Apicomplexa: Eimeriidae) in an Australian Pied Oystercatcher (Haematopus longirostris) (Aves: Charadriiformes).

A novel Eimeria Schneider, 1875 species is described from an Australian pied oystercatcher Haematopus longirostris Vieillot, in Western Australia. The pied oystercatcher was admitted to the Kanyana Wildlife Rehabilitation Centre (KWRC), Perth, Western Australia in a poor body condition, abrasion to its right hock and signs of partial delamination to its lower beak. Investigation into potential medical causes resulted in a faecal sample being collected and screened for gastrointestinal parasites. Unsporulated coccidian oocysts were initially observed in the faeces and identified as Eimeria upon sporulation. The sporulated oocysts (n = 20) are ellipsoidal, 20-21 × 12-13 µm in shape and have thick bi-layered walls which are c.2/3 of the total thickness. Micropyle is present, robust and protruding, and occasionally has a rounded polar body attached to the micropyle. Within the oocyst, a residuum, in addition, two to five polar granules are present. There are four ellipsoidal sporocysts 9-11 × 5-6 µm with flattened to half-moon shaped Stieda bodies. Sub-Stieda body and para-Stieda body are absent. The sporocysts contain sporocyst residuums composed of a few spherules scattered among the sporozoites. Within the sporozoites, anterior and posterior refractile bodies are present, but the nucleus is indiscernible. To further characterise the novel Eimeria species from H. longirostris, molecular analysis was conducted at the 18S ribosomal RNA locus, using PCR amplification and cloning. Two cloned sequences from the novel Eimeria were compared with those from other Eimeria spp. with the highest genetic similarity of 97.6% and 97.2% from Clone 1 and 2, respectively with Eimeria reichenowi (AB544308) from a hooded crane (Grus monacha Temminck) in Japan. Both sequences grouped in a clade with the Eimeria spp. isolated from wetland birds, which include Eimeria paludosa (KJ767187) from a dusky moorhen (Gallinula tenebrosa Gould) in Western Australia, Eimeria reichenowi (AB544308) and Eimeria gruis (AB544336) both from hooded cranes. Based on the morphological and molecular data, this Eimeria sp. is a new species of coccidian parasite and is named Eimeria haematopusi n. sp. after its host H. longirostris.

Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

Unveiling genotypic diversity of Theileria orientalis in lethal outbreaks among bovines in Karnataka, India.

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts.

MOLECULAR PHYLOGENY OF THE LEECH GENUS PONTOBDELLA (HIRUDINIDA: PISCICOLIDAE) WITH NOTES ON PONTOBDELLA CALIFORNIANA AND PONTOBDELLA MACROTHELA.

Leech specimens of the genus Pontobdella (Hirudinida: Piscicolidae) were found off the coast of the state of Oaxaca (Pacific) as well as in Veracruz and Tabasco (Gulf of Mexico), Mexico. Based on the specimens collected in Oaxaca, a redescription of Pontobdella californiana is provided, with emphasis on the differences in the reproductive organs with the original description of the species. In addition, leech cocoons assigned to P. californiana were found attached to items hauled by gillnets and studied using scanning electron microscopy and molecular approaches. Samples of Pontobdella macrothela were found in both Pacific and Atlantic oceans, representing new geographic records. The phylogenetic position of P. californiana is investigated for the first time, and with the addition of Mexican samples of both species, the phylogenetic relationships within Pontobdella are reinvestigated. Parsimony and maximum-likelihood phylogenetic analysis were based on mitochondrial (cytochrome oxidase subunit I [COI] and 12S rRNA) and nuclear (18S rRNA and 28S rRNA) DNA sequences. Based on our results, we confirm the monophyly of Pontobdella and the pantropical distribution of P. macrothela with a new record in the Tropical Eastern Pacific.

Redescription and molecular phylogeny of the freshwater metopid, Castula strelkowi (Jankowski, 1964) from the Czech Republic and synonymization of Pileometopus with Castula.

The relationships of the mainly free living, obligately anaerobic ciliated protists belonging to order Metopida continue to be clarified and now comprise three families: Metopidae, Tropidoatractidae, and Apometopidae. The most species-rich genus of the Metopidae, Metopus has undergone considerable subdivision into new genera in recent years as more taxa are characterized by modern morphologic and molecular methods. The genus, Castula, was established to accommodate setae-bearing species previously assigned to Metopus: C. setosa and C. fusca, and one new species, C. flexibilis. Another new species, C. specialis, has been added since. Here we redescribe another species previously included in Metopus, using morphologic and molecular methods, and transfer it to Castula as C. strelkowi n. comb. (original combination Metopus strelkowi). We also reassess the monotypic genus, Pileometopus, which nests within the strongly supported Castula clade in 18S rRNA gene trees and conclude that it represents a morphologically divergent species of Castula.

Redescription and molecular characterization of Loxocephalus luridus Eberhard, 1862 based on Czech populations: Implications for order Loxocephalida Jankowski, 1980.

The phylogenetic and taxonomic affinities of lineages currently assigned to the non-monophyletic ciliate order Loxocephalida Jankowski (1980) within subclass Scuticociliatia Small (1967) remain unresolved. In the current study, we redescribe the morphology of the type species, Loxocephalus luridus Eberhard (1862) based on two Czech populations and include the first scanning and transmission electron microscopy images of the species. We provide the first 18S rRNA gene sequences for L. luridus and consider its phylogenetic position. Our results support the separation of Dexiotricha from Loxocephalus; however, the former genus is recovered as non-monophyletic. The monophyly of genus Dexiotricha and that of Loxocephalus + Dexiotricha is rejected. Loxocephalus luridus, together with Dexiotricha species, nests within a fully supported clade with Conchophthirus species, long presumed to belong to the Pleuronematida. Haptophrya is recovered as sister to this clade. The monophyly of the Astomatia Schewiakoff (1896) including Haptophrya is rejected. No clear morphologic synapomorphy is identified for the fully supported clade consisting of Haptophrya, Dexiotricha, Loxocephalus, and Conchophthirus.

Arctic cyanobacterial mat community diversity decreases with latitude across the Canadian Arctic.

Cyanobacterial mats are commonly reported as hotspots of microbial diversity across polar environments. These thick, multilayered microbial communities provide a refuge from extreme environmental conditions, with many species able to grow and coexist despite the low allochthonous nutrient inputs. The visibly dominant phototrophic biomass is dependent on internal nutrient recycling by heterotrophic organisms within the mats; however, the specific contribution of heterotrophic protists remains little explored. In this study, mat community diversity was examined along a latitudinal gradient (55-83°N), spanning subarctic taiga, tundra, polar desert, and the High Arctic ice shelves. The prokaryotic and eukaryotic communities were targeted, respectively, by V4 16S ribosomal RNA (rRNA) and V9 18S rRNA gene amplicon high-throughput sequencing. Prokaryotic and eukaryotic richness decreased, in tandem with decreasing temperatures and shorter seasons of light availability, from the subarctic to the High Arctic. Taxonomy-based annotation of the protist community revealed diverse phototrophic, mixotrophic, and heterotrophic genera in all mat communities, with fewer parasitic taxa in High Arctic communities. Co-occurrence network analysis identified greater heterogeneity in eukaryotic than prokaryotic community structure among cyanobacterial mats across the Canadian Arctic. Our findings highlight the sensitivity of microbial eukaryotes to environmental gradients across northern high latitudes.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

Dnttip2 is one of the components of the small subunit (SSU) processome. In yeast, depletion of dnttip2 leads to an inefficient processing of pre-rRNA and a decrease in synthesis of the mature 18S rRNA. However, the biological roles of Dnttip2 in higher organisms are poorly defined. In this study, we demonstrate that dnttip2 is a maternal gene in zebrafish. Depletion of Dnttip2 leads to embryonic lethal with severe digestive organs hypoplasia. The loss of function of Dnttip2 also leads to partial defects in cleavage at the A0-site and E-site during 18S rRNA processing. In conclusion, Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

In silico assessment of 18S rDNA metabarcoding markers for the characterization of nematode communities.

Nematodes are keystone actors of soil, freshwater and marine ecosystems, but the complexity of morphological identification has limited broad-scale monitoring of nematode biodiversity. DNA metabarcoding is increasingly used to assess nematode diversity but requires universal primers with high taxonomic coverage and high taxonomic resolution. Several primers have been proposed for the metabarcoding of nematode diversity, many of which target the 18S rRNA gene. In silico analyses have a great potential to assess key parameters of primers, including taxonomic coverage, resolution and specificity. Based on a recently-available reference database, we tested in silico the performance of fourteen commonly used and one newly optimized primer for nematode metabarcoding. Most primers showed very good coverage, amplifying most of the sequences in the reference database, while four markers showed limited coverage. All primers showed good taxonomic resolution. Resolution was particularly good if the aim was the identification of higher-level taxa, such as genera or families. Overall, species-level resolution was higher for primers amplifying long fragments. None of the primers was highly specific for nematodes as, despite some variation, they all amplified a large number of other eukaryotes. Differences in performance across primers highlight the complexity of the choice of markers appropriate for the metabarcoding of nematodes, which depends on a trade-off between taxonomic resolution and the length of amplified fragments. Our in silico analyses provide new insights for the identification of the most appropriate primers, depending on the study goals and the origin of DNA samples. This represents an essential step to design and optimize metabarcoding studies assessing nematode diversity.

First report of Sarcocystis falcatula in naturally infected Razorbill auks (Alca torda) collected in Tunisian Mediterranean Sea shores.

Sarcocystis spp. are apicomplexan cyst-forming parasites that can infect numerous vertebrates, including birds. Sarcosporidiosis infection was investigated in three muscles (breast, right and left thigh muscle) and one organ (heart) of four Razorbill auks (Alca torda) stranded between November and December 2022 on the shores of the Mediterranean Sea in Nabeul and Bizerte governorates, Northern Tunisia. Two of the four tested A. torda were PCR positive for 18S rRNA Sarcocystis spp. gene. Among the examined 16 muscles/organs, only one breast and one right thigh were Sarcocystis spp. PCR-positive (12.5% ± 8.3, 2/16). Our results showed a relatively high molecular prevalence of Sarcocystis spp. in Razorbill auks (A. torda). Sarcocystis spp. sequence described in the present study (GenBank number: OR516818) showed 99.56-100% identity to Sarcocystis falcatula. In conclusion, our results confirmed the infection of Razorbill auks (A. torda) by S. falcatula. Further research is needed on different migratory seabirds' species in order to identify other Sarcocystis species.