Babesia species are single-celled parasites that invade red blood cells and are transmitted by ticks. In cattle, infections such as babesiosis cause fever, anemia, and significant economic losses, while in dogs the disease can be life-threatening. Human babesiosis, though less common, can be severe in elderly or immunocompromised patients. Because symptoms often overlap with other febrile illnesses, accurate molecular detection is essential—but conventional PCR-based diagnostics typically require laboratory infrastructure, thermal cyclers, and costly reagents that are out of reach in many field and rural settings.

The research team, led by Suphaporn Paenkaew and Kittisak Buddhachat at Naresuan University, set out to address one of the biggest bottlenecks in molecular diagnostics performed outside a laboratory: getting usable DNA out of a blood sample quickly and cheaply. The group evaluated five different blood DNA extraction approaches: a commercial DNeasy kit, simple boiling, TE buffer extraction, a methanol-based method, and Chelex 100 resin. Each extract was then fed into an RPA/CRISPR-Cas12a assay targeting the cytochrome c oxidase subunit I (COI) gene of Babesia.

Three of the five methods—the commercial kit, boiling, and TE buffer extraction—produced positive results in the CRISPR assay. The researchers then focused on the two simplest candidates, TE buffer extraction and boiling, and optimized the assay using Babesia-specific primers designed against the parasite’s 18S ribosomal RNA gene. When the performance of all extraction methods was compared head-to-head, they proved comparable to the commercial kit, but the TE buffer approach stood out for its simplicity and speed, requiring essentially no specialized equipment beyond a pipette and a source of the buffer itself.

The detection chemistry behind the assay is what makes the workflow so powerful. Recombinase polymerase amplification is an isothermal technique that copies DNA at a constant temperature—typically between body temperature and 42 degrees Celsius—without the cycling demanded by conventional PCR. In this study, the team optimized RPA conditions across a range from 25 to 45 degrees Celsius and incubation times from zero to 30 minutes, establishing conditions that work rapidly with minimal instrumentation. Once amplified, the target DNA triggers CRISPR–Cas12a, an enzyme that, upon binding its guide RNA-directed target sequence, unleashes indiscriminate cutting of nearby single-stranded DNA reporters, producing a detectable fluorescent signal.

Specificity is a critical concern for any diagnostic, and the assay passed that test. The RPA/CRISPR-Cas12a system specifically detected Babesia species including Babesia vogeli, Babesia gibsoni, and Babesia ovata—pathogens responsible for canine and bovine babesiosis in tropical and subtropical regions. The guide RNA was designed against a conserved region within the 18S rRNA gene amplification segment, with the protospacer adjacent motif and primer binding regions verified through multiple sequence alignments of Babesia sequences.

Sensitivity measurements revealed a trade-off worth noting. Using DNA extracted with TE buffer, the limit of detection was 10,000 copies per reaction, whereas the commercial kit achieved 1,000 copies per reaction—a tenfold difference in analytical sensitivity. The authors attribute this gap to the relative purity of kit-extracted DNA, which undergoes binding, washing, and elution steps that remove inhibitors. Even so, the TE-based workflow remained fully capable of detecting infections in clinical samples, suggesting that for field screening the modest sensitivity loss is an acceptable price for a dramatic reduction in cost and complexity.

The real-world validation came from clinical blood samples: seven bovine and eight canine specimens were tested using both the TE buffer extraction and the commercial kit, each followed by the RPA/CRISPR-Cas12a assay. The results showed complete concordance between the two extraction approaches, with six positive samples detected. The canine samples were additionally characterized by qPCR high-resolution melting analysis and conventional PCR with sequencing, confirming that the positives reflected genuine Babesia infections rather than assay artifacts.

The implications extend beyond veterinary clinics. The same research group has previously deployed RPA/CRISPR-Cas12a assays for other tick-borne pathogens, including Ehrlichia canis, Anaplasma platys, and Hepatozoon canis in dogs, and for Babesia vogeli specifically. Building a reliable, kit-free extraction step into that pipeline removes one of the last laboratory-dependent components. In regions where babesiosis is endemic and diagnostic laboratories are scarce, a workflow that needs only TE buffer, isothermal amplification reagents, and a CRISPR detection mix could be run in veterinary practices, farms, or mobile clinics.

The study, funded by the National Research Council of Thailand and Naresuan University, positions TE buffer extraction as a pragmatic bridge between sophisticated molecular diagnostics and the resource-limited settings that need them most. While the tenfold sensitivity gap relative to commercial kits may matter for very low-level infections, the complete agreement on clinical samples indicates that the simple method is fit for purpose. As CRISPR-based diagnostics continue to mature, studies like this one underscore that the humblest reagents—buffer, primers, and a guide RNA—can deliver laboratory-quality answers where they are needed fastest.

Subject of Research: A low-cost TE buffer DNA extraction method combined with RPA/CRISPR-Cas12a for rapid point-of-care detection of Babesia parasites

Article Title: A cost-effective and rapid TE-based DNA extraction for near point-of-care detection of Babesia spp. by RPA/CRISPR-Cas12a

Article References: Paenkaew, S., Sinthao, O., Thapthim, T., Mahakkaprateep, S., Cheukaw, A., Keawmanee, S., Euppayo, T., Tungtrakanpoung, R., Teapunvong, W., Nganvongpanit, K., & Buddhachat, K. (2026). A cost-effective and rapid TE-based DNA extraction for near point-of-care detection of Babesia spp. by RPA/CRISPR-Cas12a. Molecular Biology Reports, 53(1), Article 1545. https://doi.org/10.1007/s11033-026-12665-2

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12665-2

Keywords: Babesia, DNA extraction, TE buffer, CRISPR-Cas12a, recombinase polymerase amplification, point-of-care diagnostics, babesiosis, 18S rRNA, veterinary parasitology, tick-borne disease, molecular detection, isothermal amplification

The Euphrates-Tigris catchment is one of the major drainage basins of the Middle East, but decades of overexploitation have pushed the water system to its ecological limits. Iraqi waterbodies suffer from salinization driven by water losses caused by upstream dam construction, water policies during the Iraq wars and the intensifying impact of climate change on a region that already experiences minimal rainfall. Against this backdrop, diatoms, a diverse group of microalgae with silicified cell walls, serve as the world’s most widely used bioindicators of water quality, reflecting ecosystem conditions with unique sensitivity. Epiphytic diatoms, which live attached to submerged macrophytes, are particularly interesting because they interact with their living hosts in ways that can shape their own community structure.

Accurate taxonomic identification of diatoms is essential for sound biodiversity evaluation, yet classical identification by light microscopy requires highly specialized taxonomic skills and can produce discrepancies between researchers. Cryptic diversity, species that look identical under the microscope but are genetically distinct, further hinders accurate identification. In regions where literature on the local diatom flora is scarce, researchers often rely on European floras, which may force identifications into existing taxonomic concepts and overlook local variation or unique species. DNA metabarcoding, which enables the concurrent identification of numerous species from environmental samples, has become an increasingly popular alternative, but until now there has been a notable absence of such studies in Iraq.

The research team collected 47 samples during the summer of 2019, the dry season, and the winter of 2020, the rainy season, at six sites across two water bodies: the River Tigris in the city of Al-Qurnah and the River Euphrates in the city of Al-Midaina. Because no stones were available as habitats at the sampling stations, samples were taken separately from 11 different taxa of macrophytes and macroalgae, including common reed, cattail, hornwort, watermilfoil, several pondweed species, green algae of the genus Cladophora, naiad and sea aster. At least five stems and branches of each plant were shaken vigorously with distilled water to dislodge the attached diatoms, and the resulting suspensions were preserved with 70 percent alcohol for transport to the Botanic Garden and Botanical Museum at Freie Universität Berlin, where each sample was split for morphological and molecular analysis.

For the morphological analysis, samples were treated with 35 percent hydrogen peroxide to oxidize organic materials, and the cleaned diatom frustules were mounted in the high refractive index medium Naphrax. Using a Zeiss Axioplan microscope with differential interference contrast and a 100x Plan Apochromat objective, the researchers counted a minimum of 400 valves per sample to document the presence and relative abundance of each taxon, and turned to a Hitachi FE 8010 scanning electron microscope when finer details were needed. This meticulous dual-microscope approach identified 284 infrageneric taxa belonging to 59 genera, a species richness notably higher than in previous Iraqi studies. Remarkably, 46 species were recorded for the first time in the region, and 62 others could not be identified to species level and are possibly new to science, including Prestauroneis furatensis, which was newly described from the epiphytic community of the River Euphrates.

The metabarcoding analysis targeted the V4 region of the 18S rRNA gene, amplified with the DIV4 primer pair and sequenced on an Illumina MiSeq platform with 300-bp paired-end reads. After bioinformatic processing with the DADA2 pipeline, which denoises reads and infers amplicon sequence variants, and quality control with the metabaR package to remove contaminants and tag-jumps, 5,461,214 reads remained from 43 successfully amplified samples. These reads belonged to 2,953 ASVs, of which 1,454, or 49.2 percent, were classified as Bacillariophyta, the diatoms. The molecular approach recovered 54 genera and 108 species, with many ASVs assigned to the same taxa, and in most cases metabarcoding detected more taxa per sample than microscopy.

The comparison between the two methods exposed striking technical discrepancies. At the genus level, 38 genera were retrieved in both datasets, while 21 genera were identified only by morphology, including Amphipleura, Aulacoseira, Caloneis, Prestauroneis and Synedropsis, and 16 only by metabarcoding, including Campylodiscus, Chaetoceros, Eunotia, Fistulifera and Skeletonema. The incomplete taxonomic reference database for the studied sites turned out to be the major limitation of the molecular approach, as many morphologically identified species in genera such as Mastogloia and Achnanthidium are absent from any reference library. Intraspecific genetic variation, multiple rRNA operons within diatom genomes evolving at different rates, and sequencing artifacts can also generate multiple ASVs from a single species, artificially inflating molecular richness. Conversely, some abundant species identified microscopically, such as Navicula recens, Navicula rostellata and Nitzschia clausii, had no corresponding match in the metabarcoding inventories, possibly because of insufficient DNA concentrations in those samples.

Community-level statistics revealed further contrasts. Shannon diversity indices and average taxa richness were mostly higher in the metabarcoding analyses than in the morphological ones, with the highest molecular species count of 345 recorded at one winter Euphrates station and the lowest of 5 at another. Both approaches agreed that taxon richness was lowest at station E3 on the Euphrates and highest at station E1 on the same river. The non-metric multidimensional scaling plots for morphology showed a clear seasonal effect in diatom community composition, separating the dry summer from the rainy winter, and PERMANOVA confirmed statistically significant differences between the two seasons and the two rivers. In contrast, the NMDS plots for the 18SV4 analyses did not show a seasonal effect for the same samples, a discrepancy the authors attribute to gaps in the DNA barcode reference databases, particularly at the species level, which hinder accurate taxonomic assignment.

The ecological picture emerging from the morphological data is consistent with the environmental stress documented in the region. Pennate diatoms dominated the epiphytic assemblages, constituting 93.7 percent of identified species, while centric diatoms accounted for only 6.3 percent. The distribution of diatom assemblages showed a prevalence of freshwater species at 65.5 percent, followed by brackish species at 13.5 percent and marine species at 10 percent, with the remaining 11 percent being euryvalent species tolerant of a broad range of conditions. Several taxa of marine origin, including Tabularia fasciculata, Seminavis strigosa and Surirella striatula, were recorded, likely because the Qurna marsh connects directly with the Shatt Al-Arab River and receives water from the Arabian Gulf during high tides, creating a conduit for marine diatom migration to a region situated around 200 kilometers from the Gulf. Brackish diatoms such as Bacillaria paxillifera and Nitzschia filiformis reflect salinity fluctuations that intensify as less freshwater flows from the rivers and more seawater ingress from the Gulf.

The study demonstrates that combining metabarcoding with classical microscopy increases the detection and identification of diatom species and lays the foundation for understanding diatom biodiversity in this region. Even amid major advances in molecular tools for biodiversity assessment, the role of morphology in species discovery remains central. The authors recommend repeating the analysis with the rbcL marker, which has shown promise in providing higher resolution for diatom identification, and producing unialgal diatom strains from the studied sites to close the gaps in reference libraries. Such investigations are essential for illustrating the importance of sustainable ecosystem management and the conservation of water as a vital resource, both regionally and globally, in a catchment that climate change, overuse of waters and subsequent salinization have brought to the brink of ecological devastation.

Subject of Research: Diatom biodiversity in the Euphrates-Tigris rivers revealed by combined microscopy and DNA metabarcoding

Article Title: Comparison of the biodiversity of epiphytic diatoms in the Euphrates-Tigris rivers using morphological and metabarcoding analyses

Article References: Mohamad, H., Schimani, K., Al-Shaheen, M., Abarca, N., Jahn, R., Al-Handal, A., Kusber, W.-H., & Zimmermann, J. (2024). Comparison of the biodiversity of epiphytic diatoms in the Euphrates-Tigris rivers using morphological and metabarcoding analyses. Metabarcoding and Metagenomics, 8, Article e135082. https://doi.org/10.3897/mbmg.8.135082

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.135082

Keywords: diatoms, DNA metabarcoding, Euphrates, Tigris, biodiversity, bioindicators, water quality, Iraq, 18S rRNA, epiphytic algae, Comparison, epiphytic

The research team, led by scientists surveying more than one hundred forest localities across the island, assembled the first phylogeny of Sri Lankan goblin spiders. The family Oonopidae currently includes over 1,978 described species in 115 genera worldwide, and Sri Lanka harbours 45 recognised species across 13 genera, of which 38 are found nowhere else on Earth. To understand how these species relate to one another and to their global relatives, the researchers extracted DNA from ethanol-preserved leg tissue and amplified two nuclear ribosomal markers, 18S and 28S rRNA, the same genetic workhorses used in the earlier global phylogeny of the family published by Busschere and colleagues in 2014.

The fieldwork itself was a painstaking exercise in micro-hunting. Specimens were collected from Sri Lanka’s major climatic and physiographic zones, including the Central Mountain Range, the Knuckles Range, the low country wet zone and the dry zone. Leaf litter was sifted and left overnight in Winkler extractors, devices that coax out the small arthropods hiding within decaying vegetation. Preserved specimens were examined under stereomicroscopes and identified to genus using recent international revisionary works. The resulting dataset, comprising 43 Sri Lankan taxa, was then integrated into a global framework of 154 taxa representing 40 genera, with outgroup species from the families Dysderidae, Orsolobidae and Segestriidae providing evolutionary context.

The laboratory protocol demanded considerable ingenuity. Initial attempts to amplify fragments of 16S, CO1 and H3 genes were only partially successful, so the researchers settled on four primer pairs targeting three overlapping regions of 18S and a partial fragment of 28S. Polymerase chain reactions were run with carefully calibrated annealing temperatures, and older specimens required a specialised multiplex PCR kit to recover usable DNA. Purified products were Sanger sequenced in both directions, and the final concatenated alignment stretched to 2,782 base pairs. Maximum likelihood searches in IQ-TREE, using the best-fit GTR+F+I+G4 model of nucleotide substitution and 1,000 ultrafast bootstrap replicates, produced the phylogenetic trees at the heart of the study.

The headline finding is that the family Oonopidae is monophyletic, meaning all goblin spiders descend from a single common ancestor, which confirms earlier molecular work. But within that framework, several familiar genera turned out to be evolutionary patchworks rather than natural groups. The genera Brignolia, Opopaea, Aschnaoonops, Paradysderina, Unicorn and Silhouettella were all recovered as paraphyletic in at least some analyses, meaning their members are scattered across the tree rather than clustered together. This has direct taxonomic consequences: these genera, defined largely by morphological features such as the dorsal depression on the male palp, will need to be redefined or split.

The Brignolia and Opopaea story is particularly tangled. The study recovered a well-supported clade containing both genera, but neither was monophyletic within it. Brignolia ondaatjei emerged as the sister species of the Sri Lankan Opopaea spinosiscorona, while other Brignolia species grouped in distinct subclusters. Intriguingly, all Sri Lankan Brignolia and Opopaea species together were sister to Opopaea apicalis from the Galapagos, yet the Sri Lankan Opopaea formed a group entirely separate from their Australian, South American and African namesakes. The researchers conclude that both genera require morphological redefinition to match their evolutionary reality.

Not all results were disruptive. The genus Xestaspis, included in a goblin spider phylogeny for the first time, formed a well-supported clade of four Sri Lankan species, sister to the Australian Prethopalpus tropicus. Orchestina, the soft-bodied canopy jumpers with enlarged fourth femora, was confirmed as monophyletic, and the three Sri Lankan species formed a distinct clade sister to Orchestina saaristoi from the Congo. The genera Pelicinus and Escaphiella also emerged as natural groups. Meanwhile, the finding that most soft-bodied genera sit at the base of the oonopid tree supports the hypothesis that reduced body sclerotisation, the degree to which the body is hardened by plates, is an ancestral trait in the family, with heavily armoured species representing a derived condition.

Perhaps the most striking pattern is the sheer scale of microendemism. Of the 45 Sri Lankan species, 38 are endemic, and most are known from only one, two or three sites among the more than hundred localities surveyed. Brignolia ondaatjei is known only from two sites in the Badulla District at elevations between 990 and 1,280 metres. Xestaspis nuwaraeliya appears only above 1,700 metres in the Nuwara Eliya District. Aprusia vankhedei is restricted to two secondary forest patches within Kandy City itself. Some species, such as Aprusia rawanaellensis and Brignolia meemure, are known solely from their type localities. The phylogeny shows that endemic clades such as Aprusia, Brignolia and Xestaspis consist of closely related assemblages of multiple endemic species, strong evidence of recent, within-island speciation rather than repeated colonisation from the mainland.

The Sri Lankan taxa fall into five distinct clades within the global tree, reflecting a combination of multiple colonisation events and localised radiations. The researchers suggest that both ecological isolation and in situ speciation have driven this diversity, though they caution that it is premature to draw firm conclusions about origins because the Indian fauna remains poorly sampled. Goblin spiders, as short-range endemics with highly restricted distributions, may also serve as important indicators for monitoring the effects of climate change, agroforestry, urbanisation and pollution on forest habitats, since species confined to a single forest patch have nowhere to go when that patch degrades.

The study is a reminder that biodiversity hotspots operate at every scale, from charismatic megafauna down to spiders the size of a sesame seed hiding in the leaf litter. It also underscores the unfinished business of taxonomy: molecular trees are increasingly showing that genera erected on the basis of a handful of physical traits do not always correspond to true evolutionary lineages. With broader sampling of Indian and Southeast Asian taxa, and additional molecular markers, the researchers hope to fully resolve the origins and diversification of this ecologically important spider family. For now, Sri Lanka’s goblin spiders stand as a vivid example of how a small island, over deep time, can generate an extraordinary wealth of life in the smallest of packages.

Subject of Research: Molecular phylogenetics and microendemism of Sri Lankan goblin spiders (Oonopidae)

Article Title: Phylogenetic placement of Sri Lankan goblin spiders ( Araneae , Oonopidae ): integrating new taxa and taxonomic implications

Article References: Ranasinghe, U. G. S. L., & Benjamin, S. P. (2025). Phylogenetic placement of Sri Lankan goblin spiders (Araneae, Oonopidae): integrating new taxa and taxonomic implications. Evolutionary Systematics, 9(2), 259-270. https://doi.org/10.3897/evolsyst.9.168533

Image Credits: AI Generated

DOI: 10.3897/evolsyst.9.168533

Keywords: goblin spiders, Oonopidae, Sri Lanka, phylogenetics, microendemism, 18S rRNA, 28S rRNA, taxonomy, Brignolia, Opopaea, Orchestina, speciation

The research team, working with samples collected during the 2018 MANGAN expedition aboard the RV Sonne, compared two fundamentally different DNA extraction strategies for assessing benthic metazoan diversity. The first, called community DNA (ComDNA), involves physically isolating the tiny animals—meiofauna—from the sediment before extracting their DNA. The second, sedimentary environmental DNA (SedDNA), extracts DNA directly from whole sediment samples. Both approaches were applied to sediment cores collected from 19 stations in the eastern part of the BGR contract area, at water depths of roughly 4,000 to 4,100 meters.

The results were striking. ComDNA extractions yielded 2,145 metazoan operational taxonomic units (OTUs), while SedDNA recovered only 392 OTUs from the same cores. Even more remarkable, only 1.2 percent of OTUs were shared between the two methods. Of the 2,507 total OTUs detected across both strategies, 2,115 were unique to ComDNA and 362 were unique to SedDNA. This means the two approaches are not simply capturing the same community with different efficiency—they are detecting substantially different subsets of the fauna.

The statistical analyses reinforced this picture. Non-metric multidimensional scaling based on both Bray–Curtis and Jaccard distances revealed two distinct, non-overlapping benthic communities corresponding to the two extraction strategies. A pairwise PERMANOVA confirmed that extraction method had a highly significant effect on community composition (p < 0.001), while sampling location did not. OTU richness was significantly greater in the ComDNA dataset (p < 0.01), and Shannon diversity followed the same pattern (p < 0.05).

Taxonomically, both methods detected Annelida as the dominant phylum by relative read abundance—50.5 percent in ComDNA and 58.8 percent in SedDNA. However, the composition beneath that headline differed. In ComDNA, Nematoda accounted for 23.2 percent of reads, followed by Arthropoda (14.3 percent) and Nemertea (6.5 percent). In SedDNA, Arthropoda came second (19.5 percent) and Nematoda third (6.7 percent). Four phyla—Hemichordata, Loricifera, Chaetognatha and Entoprocta—were detected exclusively in ComDNA samples, likely because their small, free-swimming larval stages or soft bodies are captured by the organismal enrichment process but missed by direct sediment extraction.

The underlying reason for the gap is partly technical. ComDNA extraction concentrates the target organisms through sieving and centrifugation, reducing non-target DNA and PCR inhibitors while improving DNA yield and quality. In the ComDNA dataset, 6.29 percent of high-quality reads were assigned to metazoans, representing 75.7 percent of OTUs. In the SedDNA dataset, only 1.27 percent of reads and 25.9 percent of OTUs were metazoan. The rest of the signal came from fungi and diverse microeukaryotes, which swamp the metazoan signal when DNA is extracted from raw sediment.

Recognizing that SedDNA is far more practical for large-scale monitoring—it requires no onboard sieving, reduces contamination risk, and allows many more samples to be processed—the researchers modeled how much sediment would need to be processed to match the OTU richness achieved by ComDNA. Using random subsampling of sample combinations and fitting both linear and Michaelis–Menten models, they estimated that between 27 and 82 milliliters of sediment would be required, depending on sequencing depth and statistical approach. By comparison, the ComDNA protocol processed 217 milliliters of sediment per sample. At a sequencing depth of 100,000 reads per sample, the Michaelis–Menten model suggested approximately 38.8 milliliters would suffice.

The study also highlights the importance of sediment homogenization. Deep-sea benthic communities exhibit pronounced spatial heterogeneity, even at microhabitat scales, and meiofauna often cluster in patches rich in organic matter. Homogenizing the entire sediment volume before subsampling would help ensure that the DNA extract reflects the underlying community rather than a localized hotspot. However, this comes with a trade-off: homogenization eliminates vertical stratification, obscuring depth-dependent patterns in community structure. The authors recommend homogenizing each sediment layer separately if vertical resolution is needed.

For environmental impact assessments ahead of deep-sea mining, the message is clear. ComDNA extraction remains the more reliable strategy for establishing high-resolution biodiversity baselines, despite its greater cost and labor. SedDNA approaches, with optimized protocols—larger sample volumes, homogenization, and sequencing depths of at least 100,000 reads per sample—hold promise for scalable monitoring programs, particularly where rapid, broad-scale surveys are needed. The authors also call for further methodological research, including the evaluation of technical PCR replicates and standardized sediment processing protocols, to refine sediment-based eDNA approaches before they can be trusted for regulatory decisions in one of the planet’s last unexplored frontiers.

Subject of Research: Comparison of community DNA and sedimentary eDNA metabarcoding for deep-sea benthic biodiversity assessment

Article Title: Community DNA outperforms eDNA metabarcoding for biodiversity assessments in the Clarion–Clipperton Fracture Zone

Article References: Damm, L., Khodami, S., Rubel, V., Molari, M., Vink, A., Stoeck, T., & Martinez Arbizu, P. (2026). Community DNA outperforms eDNA metabarcoding for biodiversity assessments in the Clarion–Clipperton Fracture Zone. Metabarcoding and Metagenomics, 10, Article e173793. https://doi.org/10.3897/mbmg.10.173793

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.173793

Keywords: eDNA metabarcoding, community DNA, Clarion–Clipperton Fracture Zone, deep-sea mining, meiofauna, benthic biodiversity, polymetallic nodules, environmental DNA, DNA extraction, 18S rRNA, deep-sea monitoring, environmental impact assessment

The study focused on Lake Aydat, a shallow, nutrient-rich lake that stratifies and mixes twice a year, making it a classic model system for freshwater ecology. From November 2018 to January 2020, the team collected water from the sunlit euphotic zone at the deepest point of the lake, sampling monthly and intensifying to twice weekly during late spring. Fourteen litres of water per sample were filtered to concentrate microbial biomass, which was preserved at minus 80 degrees Celsius until DNA could be extracted. This careful, time-resolved sampling design was essential: the goal was not merely to list the species present, but to track how entire communities of algae, ciliates, fungi and parasites rose and fell across a full annual cycle.

The short-read side of the comparison used Illumina sequencing of the V4 hypervariable region of the 18S ribosomal RNA gene, a fragment of roughly 380 base pairs that has become the standard marker for surveying eukaryotic diversity. The long-read side used Pacific Biosciences Sequel II technology with circular consensus sequencing, a method that reads the same DNA molecule repeatedly to correct errors, achieving accuracy comparable to Illumina. Instead of a single hypervariable region, the long-read approach amplified nearly the entire ribosomal RNA operon — approximately 4,500 base pairs spanning the 18S gene, the internal transcribed spacer regions, the 5.8S gene and part of the 28S gene. In total, the team recovered more than 153,000 filtered long reads, from which they extracted over 20,000 full-length 18S sequences averaging about 1,232 base pairs each.

Both datasets were processed with comparable pipelines, clustering sequences into operational taxonomic units at 97 percent identity and assigning taxonomy through phylogenetic placement against curated reference databases. After filtering, the short-read dataset contained 747 OTUs and the long-read dataset 546. To make a rigorous, like-for-like comparison, the researchers performed an elegant analytical trick: they extracted the V4 region computationally from the long-read sequences, creating a hybrid dataset that could be clustered together with the genuine short-read OTUs. This allowed them to ask precisely whether differences in taxonomic assignment arose from the length of the sequence itself or from the different organisms each method happened to capture.

The answer was unambiguous. At broad taxonomic levels — supergroups and divisions — the two methods agreed well, painting similar pictures of the lake’s major eukaryotic lineages. But at finer scales the approaches diverged sharply. Among clusters containing sequences from both datasets, more than a quarter showed deeper taxonomic assignment with the long-read data, in some cases resolving up to seven additional hierarchical ranks. Overall, nearly 30 percent of long-read OTUs could be classified to genus level, compared with only 11 percent of short-read OTUs. The long-read dataset also recovered 35 genera that the short-read approach missed entirely, against just 13 genera unique to the short reads — and those short-read-only sequences tended to be rare, low-abundance taxa, consistent with Illumina’s greater sequencing depth.

Certain groups benefited disproportionately from the longer sequences. Ciliates, single-celled predators that are central to aquatic food webs, were frequently assigned only to phylum by short reads, while long reads resolved 34 percent of ciliate OTUs to genus. Chytrid fungi, parasites of phytoplankton with major ecological influence, saw genus-level identification jump from 8 percent with short reads to 36 percent with long reads. Diatoms, the silica-shelled algae that underpin much of freshwater primary production, were classified to genus in 92 percent of long-read OTUs versus 56 percent of short-read OTUs. Where the short-read data suggested a lake dominated by the diatom genus Fragilaria, the long-read data revealed substantial populations of Cyclotella and Asterionella — a finding that matched earlier microscopic observations of the lake, underscoring how short-read surveys can misrepresent community composition.

The seasonal dynamics the long-read data revealed are where the study becomes genuinely vivid. Because taxa could be identified precisely, the researchers could watch individual genera succeed one another through the year. The ciliate Condylostoma peaked in June 2019, while Carchesium, a related filter-feeder with similar dietary preferences, showed a different temporal pattern — a succession that may reflect differing life-history strategies or tolerances to temperature, oxygen and nutrient fluctuations. Among the diatoms, Cyclotella flourished in spring, Fragilariforma dominated in July, and Asterionella reached its greatest abundance in the cold months of January and April. Such fine-grained temporal partitioning among close relatives is exactly the kind of ecological process that short-read data, with its blurred taxonomy, tends to obscure.

Perhaps the most ecologically compelling result concerns parasitism. Chytrid fungi infect and lyse phytoplankton, sometimes terminating algal blooms and reshaping entire food webs, yet host–parasite dynamics are notoriously difficult to observe in nature. The long-read data allowed the team to track the chytrid genus Zygorhizidium alongside its diatom hosts month by month. Zygorhizidium abundance rose sharply between March and May, coinciding with the spring diatom bloom — a pattern consistent with laboratory work showing that Zygorhizidium affluens infects the diatom Asterionella formosa, but now observable directly in a natural lake. A December peak in chytrids followed a November surge of the golden alga Mallomonas, echoing previous reports of chytrid-driven algal declines. The data also showed simultaneous peaks of Perkinsea parasites and colonial green algae, supporting emerging evidence that these parasites, better known from marine hosts, play a similar role in freshwaters.

The authors are careful to note that short-read sequencing retains real advantages: it is cheaper, generates far greater depth, benefits from mature analytical pipelines, and excels at detecting rare taxa. Long reads also demand higher-quality DNA, since degraded environmental samples can fail to amplify across 4,500 base pairs, and reference databases remain thinner for full-length sequences, which occasionally forced more conservative assignments. Indeed, in a small minority of cases the short reads achieved finer classification, likely because the V4 region is better represented in reference databases. These caveats matter for anyone designing a monitoring programme, and the study does not suggest that short reads should be abandoned.

But the trajectory is clear. As sequencing costs fall and reference databases grow, the study argues that long-read metabarcoding should move from specialist tool to routine instrument of environmental surveillance. The ability to resolve genera and species in time series opens the door to tracking bioindicator ciliates for water quality, detecting parasitic outbreaks before blooms collapse, and mapping the cryptic diversity that short fragments cannot distinguish. Combined with the 28S sequences the team plans to analyse next, and with emerging single-cell and genome-based reference collections, long-read surveys promise to close taxonomic gaps that have limited freshwater ecology for two decades. For the invisible majority of lake life, the picture is finally coming into focus — one long read at a time.

Subject of Research: Comparing short-read and long-read metabarcoding of eukaryotic diversity in a lake over one year

Article Title: Profiling the eukaryotic diversity over a year in a lake ecosystem through short- and long-read metabarcoding

Article References: Chauvet, M., Terzian, P., Moné, A., Debroas, D., Bronner, G., & Lepère, C. (2026). Profiling the eukaryotic diversity over a year in a lake ecosystem through short- and long-read metabarcoding. Metabarcoding and Metagenomics, 10, Article e184566. https://doi.org/10.3897/mbmg.10.184566

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.184566

Keywords: metabarcoding, long-read sequencing, PacBio, eukaryotic diversity, Lake Aydat, 18S rRNA, chytrid fungi, diatoms, ciliates, freshwater ecology, seasonal dynamics, host-parasite interactions

The study, published in PLOS Water, was led by Justin Gibbons, Heidi Pagán, Tatiana Ferrer, Rays H. Y. Jiang, and Greg O’Corry-Crowe. Rather than relying on traditional microscopy or conventional short-read genetic sequencing, the team turned to Oxford Nanopore Technologies’ MinION platform, a device that reads long strands of DNA by threading them through tiny protein pores and measuring the electrical signals produced as each base passes through. This long-read approach allowed the researchers to target the 18S rRNA gene, a genetic marker specific to eukaryotic organisms, the branch of life that includes algae, dinoflagellates, and other complex single-celled organisms responsible for many of the world’s most damaging blooms.

The choice of target matters enormously. Most environmental DNA surveys of aquatic microbial communities have historically focused on the 16S rRNA gene, which preferentially captures bacteria and archaea, the prokaryotic members of the microbial world. While these surveys are valuable for tracking bacterial communities, they largely miss the eukaryotic species that form harmful algal blooms. By designing their survey around the 18S marker and pairing it with long-read sequencing, the researchers were able to generate community profiles that resolved eukaryotic diversity at a level of detail that shorter reads and prokaryote-focused assays simply cannot match.

Sampling was ambitious in scope. The team collected water from 20 sites spread across the lagoon, capturing both wet and dry seasons and covering the estuary’s full latitudinal range, which spans both subtropical and temperate subregions. This design allowed them to ask not just which harmful species are present, but where and when they flourish, and how their distributions track the environmental conditions of the lagoon. The Indian River Lagoon is a notoriously complex system, with restricted ocean inlets, variable salinity gradients, and nutrient inputs from a heavily developed watershed, all of which shape the microbial communities living in its waters.

The sequencing results were striking. Across the samples, the team detected more than 200 unique eukaryotic species, and the community composition differed clearly by season, by region, and along environmental gradients including salinity, temperature, and nutrient ratios. These patterns offer a window into the ecological forces that govern which microbes dominate at any given place and time. In an estuary where conditions can shift rapidly with rainfall, runoff, and temperature, understanding these relationships is essential for predicting when conditions become favorable for a bloom to ignite.

Most importantly from a public health and ecological standpoint, the survey detected eleven harmful algal bloom taxa, including some of the most notorious offenders in Florida waters. Among them were Aureoumbra lagunensis, the golden alga responsible for the devastating brown tide events that have clouded lagoon waters and collapsed seagrass meadows; Karlodinium veneficum, a dinoflagellate that produces ichthyotoxins capable of killing fish; and Pyrodinium bahamense, which produces saxitoxins that accumulate in shellfish and can cause paralytic shellfish poisoning in humans. In some sampling sites, Aureoumbra lagunensis accounted for more than 50 percent of the relative abundance of the eukaryotic microbial community, a dominance that signals the kind of near-monoculture conditions characteristic of an active or incipient brown tide.

The ability to detect such dominance with genetic tools rather than microscopy carries practical advantages. Traditional bloom monitoring depends heavily on expert taxonomists identifying organisms under a microscope, a process that is labor-intensive, slow, and dependent on the availability of specialized skills. Morphological identification can also be ambiguous, since closely related species may look nearly identical while differing sharply in toxicity. Genetic barcoding of the 18S region sidesteps many of these problems, offering a molecular fingerprint that can distinguish species even when their physical forms cannot be told apart.

What sets the nanopore approach apart from earlier genetic methods is its potential for near real-time, field-deployable analysis. The MinION sequencer is compact and can be run outside a conventional laboratory, meaning that water samples collected from the lagoon could in principle be processed on-site or nearby, with results available in hours rather than days or weeks. For resource managers charged with protecting public health, closing shellfish beds, or warning recreational users, that speed could make the difference between a timely intervention and a costly after-the-fact response. The authors present the approach as a promising tool for rapid detection of harmful algal bloom species in complex estuarine systems like the Indian River Lagoon.

The findings also carry broader scientific significance. By documenting how eukaryotic microbial communities shift across seasons, regions, and environmental gradients, the study improves understanding of the ecological drivers that shape bloom risk. Salinity, temperature, and the ratios of nutrients such as nitrogen and phosphorus are known to influence which species can thrive, and the lagoon’s north-to-south span across climatic zones makes it a natural laboratory for studying how these factors interact. As climate change alters rainfall patterns, warms coastal waters, and intensifies nutrient runoff in estuaries worldwide, tools that can track microbial communities at fine spatial and temporal resolution will become increasingly valuable for anticipating, rather than merely reacting to, bloom events.

The Indian River Lagoon itself has become a symbol of both ecological vulnerability and scientific urgency. Its waters support dolphins, manatees, sea turtles, and thousands of other species, and its seagrass beds, already stressed by decades of blooms and nutrient loading, form the foundation of the estuary’s food web. Monitoring programs that can rapidly identify which harmful species are present, and in what abundance, give scientists and managers a fighting chance to protect these resources. This study demonstrates that long-read sequencing of the 18S rRNA gene can capture the full breadth of eukaryotic diversity in estuarine waters while resolving the potentially harmful taxa that matter most, offering a template that other coastal systems, from Chesapeake Bay to the Gulf of Mexico, could readily adopt as harmful algal blooms continue to intensify around the world.

Subject of Research: Near real-time long-read eDNA sequencing of 18S rRNA to monitor harmful algal bloom species in the Indian River Lagoon estuary

Article Title: Near real-time long-read eDNA sequencing reveals harmful algal bloom dynamics in the Indian River Lagoon

Article References: Gibbons, J., Pagán, H., Ferrer, T., Jiang, R. H. Y., & O’Corry-Crowe, G. (2026). Near real-time long-read eDNA sequencing reveals harmful algal bloom dynamics in the Indian River Lagoon. PLOS Water, 5(10), e0000557. https://doi.org/10.1371/journal.pwat.0000557

Image Credits: AI Generated

DOI: 10.1371/journal.pwat.0000557

Keywords: harmful algal blooms, environmental DNA, nanopore sequencing, Indian River Lagoon, 18S rRNA, Aureoumbra lagunensis, Karlodinium veneficum, Pyrodinium bahamense, estuarine ecology, brown tide, water quality monitoring, Oxford Nanopore MinION

The scale of the fieldwork gives the results unusual weight. The team collected 3,151 ticks in total from 805 cattle and 95 buffaloes. The collection was dominated by adult females, which accounted for 1,630 individuals or 51.7 percent of the haul, followed by 725 males at 23 percent, 458 nymphs at 14.5 percent and 338 larvae at 10.7 percent. This stage structure matters because different life stages feed on hosts at different times of year and differ in their capacity to acquire and transmit pathogens. Alongside the tick collections, the researchers drew 221 blood samples from animals that were both showing clinical signs and carrying ticks, a deliberately targeted sampling strategy designed to capture active infection rather than background carriage.

Morphological identification of the ticks revealed a two-genus community. Rhipicephalus species made up 83.59 percent of the collection, with Hyalomma accounting for the remaining 16.41 percent. Within Rhipicephalus, the cattle tick Rhipicephalus microplus was overwhelmingly dominant at 70.29 percent of all ticks collected, followed by Rhipicephalus haemaphysaloides at 7.61 percent and an unidentified Rhipicephalus at 5.68 percent. The Hyalomma contingent comprised Hyalomma anatolicum at 8.82 percent and Hyalomma turanicum at 7.58 percent. The dominance of R. microplus is significant for regional biosecurity, because this one-host tick is notorious worldwide for developing resistance to common acaricides and for its efficiency as a vector of bovine babesiosis and anaplasmosis, in addition to its role in theileriosis transmission dynamics.

Infestation patterns differed sharply between host species. Cattle carried ticks at a rate of 46.33 percent, more than double the 22.11 percent recorded in buffaloes. Among cattle, the three districts were remarkably similar, with infestation rates of 46.49 percent in Upper Dir, 46.62 percent in Bajaur and 45.90 percent in Lower Dir, suggesting that the burden is region-wide rather than localized. Buffaloes showed correspondingly lower figures of 24.14, 23.53 and 18.75 percent in the same districts. Statistical analysis confirmed that host species itself was a significant determinant of infestation, a finding consistent with known differences in grooming behavior, skin thickness and husbandry between the two ruminants.

The diagnostic comparison at the heart of the study illustrates why microscopy alone can seriously underestimate tick-borne infection. Conventional blood-smear examination detected Theileria in 18.09 percent of the 221 samples, that is 40 animals, comprising 37 of 202 cattle at 18.31 percent and 3 of 19 buffaloes at 15.78 percent. When the same samples were screened by polymerase chain reaction targeting the parasite’s 18S ribosomal RNA gene, the detection rate jumped to 45.24 percent, with 100 of 221 animals positive, including 92 cattle at 45.54 percent and 8 buffaloes at 42.11 percent. The roughly two-and-a-half-fold gap between the two methods reflects the limited sensitivity of microscopy, which depends on the density of parasites in peripheral blood and the skill of the microscopist, whereas PCR can amplify parasite DNA even at very low parasitemia and in carrier animals that appear clinically recovered.

To identify the parasite species, the researchers sequenced the amplified 18S rRNA fragment and compared it against public databases using BLAST analysis. The sequence showed a maximum identity of 99.62 percent with Theileria annulata reference sequences from Turkey, China and Pakistan, and a phylogenetic tree constructed in MEGA X using the maximum-likelihood method placed the Pakistani isolate firmly within a clade containing T. annulata sequences from Asia and Europe. The 18S rRNA gene is a standard barcode for piroplasmid parasites because it is conserved enough for reliable species-level discrimination yet variable enough to resolve relationships. The finding confirms that the causative agent of tropical theileriosis, one of the most economically damaging tick-borne diseases of cattle across North Africa, the Middle East and South Asia, is the species circulating in northern Pakistan. The consensus sequence has been deposited in GenBank under accession number PV538505, making it available for future comparative studies.

The risk-factor analysis produced a nuanced picture of what drives infestation and infection in these herds. Age, season and acaricide treatment were significantly associated with tick infestation in both cattle and buffaloes, with p values below 0.05. Younger animals and untreated animals carried higher burdens, and tick numbers rose and fell with the seasons in line with the activity patterns of the vector species. For Theileria infection itself, age, season and acaricide use were significant in cattle, whereas in buffaloes only acaricide use reached statistical significance. This asymmetry suggests that buffaloes, with their lower infestation rates, may experience infection dynamics that depend more heavily on chemical tick control than on the demographic and seasonal factors that dominate in cattle.

Equally instructive are the factors that did not matter. Gender, the presence of rural poultry, the grazing system and the district of origin showed no significant association with either tick infestation or Theileria infection in either host species. The uniformity across the three districts reinforces the conclusion that the parasite and its vectors are well established throughout the region. The irrelevance of poultry presence and grazing system suggests that interventions targeting those variables alone would be unlikely to reduce disease pressure, sharpening the focus on the factors that do matter.

The practical implications of the study are considerable for a livestock-dependent economy. Tropical theileriosis caused by T. annulata produces fever, anemia, lymph node swelling and, in severe cases, death in cattle, and even subclinical infections depress milk yield and weight gain. Studies from North Africa have documented substantial milk losses attributable to the disease, and comparable costs are plausible in Pakistani production systems where dairy output is a primary household income source. The identification of acaricide treatment as a significant protective factor points to the central role of tick control, but it also raises a warning: R. microplus is among the tick species most prone to acaricide resistance globally, so indiscriminate chemical use could erode the very tool that the data show to be effective. The authors suggest that their integrated assessment, linking pathogen, vector and risk factors from the same infected hosts, can help guide preventive measures in the mountainous terrain of northern Pakistan.

Methodologically, the study demonstrates the value of integration. By pairing morphological tick taxonomy with molecular pathogen detection and phylogenetics on samples drawn from the same animals, the researchers avoided the fragmentation that has limited many earlier surveys, which examined either vectors or parasites in isolation. The work also contributes to a growing body of Pakistani tick research, including recent systematic reviews of Hyalomma anatolicum and Rhipicephalus microplus distribution and updated national checklists of tick fauna. As climate change alters the distribution patterns of ticks and the pathogens they carry, baseline datasets of this kind become essential reference points. For farmers in Upper Dir, Lower Dir and Bajaur, the message is concrete: seasonal tick control, particularly for young cattle, remains the most actionable defense against a disease whose molecular fingerprint now places it squarely within the well-known Asian and European lineage of Theileria annulata.

Subject of Research: Molecular epidemiology of Theileria annulata infection and tick infestation in cattle and buffaloes in northern Pakistan

Article Title: Molecular Epidemiology and Risk Factors of Theileria annulata Infection and Rhipicephalus and Hyalomma Tick Infestation in Three Districts of Northern Pakistan

Article References: Rahman, Z. U., Khan, A., Wasim, M., Wahid, S., Gul, M., Khan, M., Aljasham, A. T., & Ali, A. (2026). Molecular Epidemiology and Risk Factors of Theileria annulata Infection and Rhipicephalus and Hyalomma Tick Infestation in Three Districts of Northern Pakistan. Acta Parasitologica, 71(5), Article 237. https://doi.org/10.1007/s11686-026-01416-9

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01416-9

Keywords: Theileria annulata, tropical theileriosis, Rhipicephalus microplus, Hyalomma anatolicum, 18S rRNA, PCR, phylogenetic analysis, risk factors, livestock, Pakistan, tick-borne disease, cattle

The research team, led by Huan Li and Lei Ma of Hebei Normal University together with Jing Huang of Hebei Children’s Hospital, assembled an unusually large sample set for this kind of survey: 2,448 human fecal specimens and 215 ticks of the species Haemaphysalis longicornis, an aggressive, wide-ranging biting tick common across East Asia. Using polymerase chain reaction assays designed to amplify the 18S rRNA gene, a standard molecular marker for identifying eukaryotic microorganisms, the researchers screened every sample for Colpodella DNA. The 18S rRNA gene is favored in studies of protist diversity because it is present in all eukaryotes, contains both conserved and variable regions, and allows newly recovered sequences to be compared against a deep archive of reference sequences from known organisms.

The results were striking in their consistency across the two very different sample types. Colpodella DNA was detected in 136 of the human fecal samples, a detection rate of 5.56 percent, and in 11 of the tick samples, a rate of 5.12 percent. The near-identical prevalence in feces and ticks is one of the more intriguing aspects of the study, hinting at some shared route of exposure or a common environmental reservoir linking the human and tick samples, though the design of the study cannot distinguish among the many possible explanations, including ingestion of contaminated food or water, environmental pass-through, or a genuine biological association with the tick vector.

Every PCR-positive sample yielded a high-quality 18S rRNA sequence when the amplified DNA was read by Sanger sequencing, a validation step that strengthens confidence in the specificity of the detection. Because some of the tick-derived sequences were identical to one another, the researchers collapsed the data into representative sequence types, ultimately selecting one tick-derived sequence and 21 distinct human-derived sequence types for deeper analysis. These 22 representative sequences were deposited in GenBank under accession numbers QB017305 through QB017326, making them available to other researchers for comparison and future surveillance work.

Phylogenetic analysis, the technique biologists use to place newly recovered gene sequences on the tree of life, showed that all 22 representative sequences from the study clustered within previously reported Colpodella lineages. In other words, the DNA found in human stool and ticks belongs to the same evolutionary neighborhood as Colpodella species that have been described from aquatic and terrestrial habitats around the world. The sequences did not branch off into entirely novel territory, which suggests that the organisms detected in Hebei are related to known forms rather than representing a radically divergent new lineage, although species-level identification in this group remains difficult because morphology and molecular data do not always align neatly.

To probe the genetic variation within their dataset, the team performed a haplotype analysis, a method that groups sequences by their shared sets of variable sites. Among the analyzed sequences, they identified three haplotypes, indicating a modest but real degree of genetic diversity among the Colpodella organisms whose DNA was recovered from the samples. Haplotype networks and diversity statistics are standard tools in population genetics, and applying them to environmental protist DNA can reveal whether a detection reflects a single widespread genotype or a mixture of related strains circulating in the environment. In this case, the limited number of haplotypes suggests relatively constrained diversity in the sampled region, though the authors caution that broader sampling would be needed to draw firmer conclusions.

Understanding why this matters requires a short detour into evolutionary biology. Colpodella species are predatory flagellates that feed by a process called myzocytosis, in which they attach to another microbe, puncture its membrane, and suck out its cytoplasmic contents. This dramatic feeding strategy has fascinated cell biologists in part because it may resemble the evolutionary steps that gave rise to intracellular parasitism. Colpodellids are considered the free-living sister group to the Apicomplexa, the phylum that includes Plasmodium, the malaria parasite, and Toxoplasma gondii. Studying Colpodella therefore offers a window into how some of humanity’s most burdensome parasites may have evolved from free-living ancestors that once hunted their prey from the outside.

The new study also fits into a rapidly expanding literature on where Colpodella DNA shows up outside of ponds and soil. Recent surveys have reported the organism’s genetic traces in goats, foxes, and birds; in Hyalomma ticks infesting camels in Egypt; in ticks feeding on ruminants in Italy; in horses; in zoo animals; and even in the urine of a human patient. Clinically, isolated case reports have described Colpodella-like parasites in a woman in China and in a patient with neurological symptoms whose infection was linked to a potential novel tick-borne Colpodella species. A 2025 scoping review of the genus’s biological characteristics and zoonotic potential underscored how much remains unknown about whether these detections represent transient environmental exposure, colonization, or true infection.

That uncertainty is central to how the Hebei findings should be interpreted, and the authors are explicit about the limits of their method. PCR-based detection confirms only that Colpodella DNA was present in the sample; it cannot determine whether live organisms were present, whether they were actively replicating, whether they colonized the human gut or the tick, or whether they caused any disease. DNA can persist after an organism has died, and environmental contamination or passive passage through the body can both produce positive signals. Distinguishing among these possibilities will require complementary approaches, including attempts to culture the organisms, sequencing of additional genetic markers beyond the 18S rRNA gene, and biological studies of how Colpodella interacts with animal hosts.

Nevertheless, the study marks a meaningful addition to surveillance data on an understudied group of protists. The large number of human samples screened, the near-matching detection rates in feces and ticks, the successful sequencing of every positive sample, and the public deposition of 22 representative sequences collectively provide a foundation for future work. As molecular surveys continue to sweep ever more environments and host species, Colpodella keeps appearing in unexpected places, and each new detection sharpens the questions that make this genus scientifically compelling: how these predatory relatives of apicomplexan parasites move between environmental reservoirs, animals, and occasionally people, and whether any of them pose a genuine health risk. For now, the answer in Hebei Province is a well-documented molecular presence and a clear agenda for the research that must follow.

Subject of Research: Molecular detection and genetic diversity of Colpodella protist DNA in human fecal and tick samples in China

Article Title: Molecular Detection and Genetic Characterization of Colpodella spp. DNA in Human Fecal and Tick Samples from Hebei Province, China

Article References: Li, H., Huang, J., Zhang, M., Zhang, Y., Chen, Y., & Ma, L. (2026). Molecular Detection and Genetic Characterization of Colpodella spp. DNA in Human Fecal and Tick Samples from Hebei Province, China. Acta Parasitologica, 71(5), Article 234. https://doi.org/10.1007/s11686-026-01419-6

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01419-6

Keywords: Colpodella, protists, Apicomplexa, Haemaphysalis longicornis, ticks, 18S rRNA, PCR detection, genetic diversity, haplotypes, Hebei Province, zoonotic potential, myzocytosis

Cryptosporidium species are obligate intracellular apicomplexan parasites that infect the intestinal epithelium of a wide range of vertebrates, causing gastroenteritis and diarrhea that can be severe in young animals and dangerous, sometimes life-threatening, in immunocompromised people. Unlike many enteric pathogens, Cryptosporidium oocysts are remarkably resistant to environmental stress and to standard water-treatment disinfection, which makes even asymptomatic carriers relevant to public health. Because infected animals shed infectious oocysts in their feces without necessarily showing signs of disease, understanding which species inhabit which hosts is central to assessing transmission risk on farms and in watersheds.

The research team, led by Sümmani Çifci of Aksaray University together with colleagues at Erciyes University, focused on the Akkaraman breed, a hardy indigenous sheep breed that dominates the flocks of Central Anatolia. Between the provinces of Nevşehir, Sivas, and Kayseri, the investigators collected fresh fecal samples from 300 healthy Akkaraman sheep, one hundred animals per province, drawn randomly from a variety of small-scale farms. Restricting the survey to clinically healthy animals was a deliberate choice: it allowed the team to measure the burden of subclinical infections, the silent reservoir that conventional disease surveillance tends to miss.

In the laboratory, genomic DNA was extracted from each sample and screened for Cryptosporidium using polymerase chain reaction (PCR), a technique that amplifies parasite-specific DNA sequences to levels that can be detected even when oocyst numbers are low. Positive samples then underwent sequence analysis of the 18S ribosomal RNA gene, the standard genetic barcode for distinguishing Cryptosporidium species, whose oocysts are morphologically nearly identical under the microscope. The resulting sequences were compared against reference databases and used to build phylogenetic trees, placing each isolate within the parasite’s evolutionary framework.

The headline result was an overall prevalence of 6.0 percent, with 18 of the 300 sheep testing positive for Cryptosporidium. Sequence analysis resolved these infections into three species: Cryptosporidium parvum and Cryptosporidium bovis each accounted for seven animals, corresponding to 2.3 percent of the flock sample, while Cryptosporidium xiaoi was found in four animals, or 1.3 percent. The team deposited the sequences in the GenBank database under accession numbers MZ027074 through MZ027078, making them available for future comparative studies worldwide.

The detection of C. bovis in sheep is the study’s most striking claim. As its name suggests, C. bovis is primarily a cattle parasite, typically associated with subclinical infections in post-weaned calves and adult bovines. Finding it in ovine hosts in Türkiye is, according to the authors, a first for the country and adds to a growing international literature showing that host boundaries among Cryptosporidium species are more permeable than once assumed. Such cross-species spillover matters because it hints at possible transmission between cattle and sheep sharing pasture, water sources, or farm infrastructure, and because it complicates the simple assumption that each livestock species carries its own characteristic parasite fauna.

The other two species found carry their own significance. C. parvum is the most clinically important Cryptosporidium in mammals and a major zoonotic agent, responsible for a large share of human cryptosporidiosis cases worldwide, including outbreaks linked to contaminated drinking water. Its presence in apparently healthy ewes and rams underscores that even symptom-free sheep can contribute oocysts to the environment. C. xiaoi, by contrast, is considered largely host-adapted to sheep and goats and is generally of lower zoonotic concern, though it has been documented in periparturient transmission from ewes to lambs. The mixture of zoonotic and host-adapted species within a single flock profile illustrates the epidemiological complexity of ovine cryptosporidiosis.

Statistical analysis of the survey data added an age dimension to the picture. Positivity rates differed significantly across age groups, with a p-value below 0.0001, indicating that the probability of detecting Cryptosporidium DNA was far from uniform among young and older animals. This pattern is consistent with the biology of the parasite: young lambs are typically more susceptible to active infection and higher oocyst shedding, while adults more often harbor transient or low-level infections. Notably, no significant difference emerged between male and female sheep, with a p-value of 0.548, suggesting that sex does not meaningfully shape infection risk in this population.

Phylogenetic analysis reinforced the coherence of the species identifications. The Anatolian isolates clustered within the same clades as reference sequences reported from different hosts and geographic regions, meaning that the Turkish parasites are genetically consistent with their counterparts elsewhere rather than representing divergent local lineages. This kind of global genetic continuity is exactly what allows molecular epidemiologists to trace potential transmission routes across farms, regions, and even countries, and it strengthens confidence that the species assignments were not artifacts of sequencing noise.

For the authors, the findings carry a clear practical message: Cryptosporidium infections in sheep deserve active control attention, not only because of their potential impact on lamb health and productivity but because of their public health relevance. Healthy sheep on small-scale farms, the study shows, can silently host both zoonotic C. parvum and the previously unrecorded bovine species C. bovis, contributing to environmental contamination that may reach humans through water, food, or direct contact. As molecular surveillance expands, the team suggests that the true diversity of Cryptosporidium circulating among Türkiye’s small ruminants is likely greater than microscopy-based surveys have revealed, and that continued genotyping will be essential for mapping and ultimately interrupting transmission at the human-animal interface.

Subject of Research: Molecular prevalence and species characterization of Cryptosporidium parasites in sheep in Central Anatolia, Türkiye

Article Title: Molecular Characterization of Cryptosporidium spp. in Sheep from Central Anatolia: First Report of Cryptosporidium bovis in Ovine Hosts in Türkiye

Article References: Çifci, S., Arslanhan, B. A., Önder, Z., & Düzlü, Ö. (2026). Molecular Characterization of Cryptosporidium spp. in Sheep from Central Anatolia: First Report of Cryptosporidium bovis in Ovine Hosts in Türkiye. Acta Parasitologica, 71(5), Article 231. https://doi.org/10.1007/s11686-026-01418-7

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01418-7

Keywords: Cryptosporidium, sheep, Akkaraman, Central Anatolia, Türkiye, Cryptosporidium bovis, Cryptosporidium parvum, Cryptosporidium xiaoi, 18S rRNA, molecular epidemiology, zoonosis, prevalence

The research, led by Habib Francis of the Sydney Clinical Trials Centre at The University of Sydney together with colleagues across Australia, Hong Kong and India, drew on plasma samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial, a landmark randomised controlled trial of nearly 10,000 adults with type 2 diabetes. Because the samples came from a large, well-characterised clinical cohort, the team could interrogate the behaviour of 18 S rRNA across hundreds of real biobanked specimens rather than a handful of laboratory controls. In total, 789 plasma RNA samples analysed across 17 separate OpenArray runs formed the backbone of the stability assessment, giving the study a statistical weight that most reference-gene validations never approach.

The technical challenge the authors set out to resolve is a genuine worry in multiplexed qPCR. Microfluidic OpenArray plates pack thousands of nanolitre-scale reactions onto a single plate, and the tiny input of plasma RNA, which is naturally dilute and dominated by fragmented extracellular species, usually forces a pre-amplification step. During those 14 cycles of targeted pre-amplification, all the primer pairs in the pool compete for the same enzymes, nucleotides and reaction space. Highly abundant transcripts such as 18 S rRNA, which can be present at levels orders of magnitude above the messenger RNA targets of interest, could in theory monopolise shared reagents and quietly suppress the amplification of scarcer targets. If that happened, Ct values would shift in ways that no amount of normalisation could correct, corrupting every downstream comparison.

To test this directly, the researchers ran a paired experiment on 23 plasma samples. Each sample was pre-amplified twice, once with a primer pool containing the 18 S rRNA assay and once with an otherwise equivalent pool lacking it. The team then measured four representative target genes, ACTG1, ACTB, CLU and PFN1, under both conditions and compared the raw cycle threshold values sample by sample. Because not every gene was detectable in every sample, the effective sample sizes ranged from eight to seventeen per assay, a limitation the authors acknowledge candidly. Even so, the paired design is the cleanest possible way to isolate the effect of a single primer from all other sources of variation.

The verdict was reassuring. Median paired Ct differences between the two pre-amplification conditions ranged from just 0.07 to 0.52 cycles, and none of the four targets showed a statistically significant shift, with all p-values exceeding 0.05. Bland–Altman analysis, which plots the difference between paired measurements against their average, confirmed the picture: the mean bias across all four genes combined was a negligible −0.18 Ct, and the data points for each gene were distributed similarly, mostly falling within the 95 percent limits of agreement. In plain terms, adding 18 S rRNA to the pre-amplification pool did not measurably distort the amplification of the co-amplified messenger RNA targets under the conditions tested. The feared competition effect, at least in this workflow, did not materialise.

The stability data were equally encouraging. Across the 789 samples and 17 runs, 18 S rRNA was consistently detectable, with a median Ct of 14.5 and an interquartile range of 13.4 to 16.3. The distribution of Ct values was unimodal, clustering tightly around the median without the bimodality or systematic shifts that would hint at batch effects or hidden technical artefacts. Inter-run variability was low, with a coefficient of variation of just 3.9 percent and no significant differences between runs. Notably, these results were obtained using archived plasma samples that had endured three freeze–thaw cycles, conditions typical of longitudinal clinical biobanking, suggesting that 18 S rRNA detection remains technically robust even when samples have been handled the way real-world studies demand.

The study also tackled a practical problem that haunts long-running biomarker projects: what happens when a primer pool runs out? The FIELD analyses had relied on an archived pre-amplification pool containing the 18 S rRNA assay, stored unopened at −80 °C for more than five years. The researchers compared 18 S rRNA Ct values in 774 paired plasma samples amplified with either that archived pool or a newly manufactured one. Amplification succeeded under both conditions, but the new pool produced slightly lower Ct values, a statistically significant shift with a mean bias of 0.53 Ct. More concerning was the spread: the standard deviation of paired differences was 3.82 Ct, with 95 percent limits of agreement stretching from −6.95 to 8.01 Ct, and variability appeared to widen at higher average Ct values, where template abundance is lowest.

That combination of a small average bias and wide individual-level variability carries an important lesson for anyone managing longitudinal studies. The authors conclude that data generated with archived and new primer pools remain broadly comparable at the group level, but the two pools should not be treated as directly interchangeable for individual paired measurements. They recommend practical safeguards: keeping detailed records of primer sequences, concentrations and manufacturing lots; running bridging experiments on a representative subset of samples whenever a pool must be replaced; and using paired comparisons with Bland–Altman analysis to quantify any systematic bias before transitioning. Where the bias is small and consistent, calibration may suffice; where variability is large, reanalysis with a common pool or an alternative validated assay may be the only defensible option.

The authors are careful to frame what this study does and does not prove. Their findings establish the technical performance of 18 S rRNA within plasma-based OpenArray workflows, not its biological stability across disease states or experimental conditions, and they cannot be assumed to extend to other sample types, platforms or pre-amplification chemistries. Plasma is a peculiar analytical matrix, rich in fragmented extracellular RNA and poor in total messenger RNA, and pre-analytical variables such as blood collection tube type can reshape extracellular RNA profiles entirely. Still, by combining large-scale stability assessment, paired technical comparisons and a real-world test of reagent longevity, the study offers a template for validating endogenous controls in any high-throughput qPCR setting, and it gives biomarker researchers working with scarce clinical plasma a solid reason to trust one of molecular biology’s oldest internal controls.

Subject of Research: Validation of 18 S rRNA as an endogenous control gene in high-throughput plasma RNA qPCR workflows

Article Title: Evaluation of 18 S rRNA as an endogenous control in OpenArray® plasma RNA qPCR workflows

Article References: Francis, H., Januszewski, A. S., Mangani, A., Ema, F. K., Hardikar, A. A., Joglekar, M. V., Huang, M. L., Sullivan, D. R., Ma, R. C., Galande, S., Gebski, V., Simes, R. J., Jenkins, A. J., Molloy, M. P., & Keech, A. C. (2026). Evaluation of 18 S rRNA as an endogenous control in OpenArray® plasma RNA qPCR workflows. Molecular Biology Reports, 53(1), Article 1641. https://doi.org/10.1007/s11033-026-12820-9

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12820-9

Keywords: qPCR, 18S rRNA, OpenArray, plasma RNA, endogenous control, pre-amplification, reference gene, Bland-Altman analysis, FIELD trial, biomarkers, gene expression normalisation, technical validation