The ITS region of nuclear ribosomal DNA serves as the standard DNA barcode for identifying fungi. Because many fungal species resist cultivation in the laboratory, researchers typically amplify and sequence this marker directly from environmental samples, a technique known as amplicon sequencing or metabarcoding. The raw reads, however, are not species themselves; they must be processed computationally into units that approximate species. OTU approaches cluster reads at a similarity cutoff, traditionally 97 percent, while ASV approaches, exemplified by the widely used DADA2 algorithm, denoise the data to recover the true underlying sequences. The two methods differ conceptually: ASVs are supposed to represent actual biological sequences and can be compared across studies, whereas OTUs are clusters of similar sequences that are specific to each study. Despite years of debate, no consensus has emerged on which approach best represents the true fungal community.

Previous studies have reached conflicting conclusions. Some work on bacterial 16S data found no difference between ASVs and OTUs at the family level, and broad-scale ecological patterns have proven robust to the choice of method for both bacterial and fungal amplicons. Yet concerns persist that fungal ASVs might inflate estimates of alpha diversity because ribosomal DNA exists in multiple copies and varies within species. Other researchers have reported the opposite, that OTUs exaggerate diversity through false positives; one analysis of a 189-species mock community identified between 577 and 1413 OTUs. Tosadori and Bosch designed their study to cut through this confusion by exploiting a key advantage unavailable in real-world experiments: perfect knowledge of the ground truth.

The team downloaded more than 1500 fungal genomes from 859 species in the Ensembl Fungi database and used them to simulate amplicon sequencing of the ITS1 and ITS2 regions. They built mock communities spanning 50 to 800 fungal genomes, with abundances following a power law distribution, and simulated sequencing errors, chimeras, and quality profiles modeled on real Illumina data. Because the true composition of each community was known exactly, any discrepancy between the analyzed results and reality could be attributed directly to the analysis pipeline. The researchers then processed these data through six pipelines combining two read-processing strategies with three ASV or OTU methods, evaluating diversity metrics, taxonomic composition, sensitivity, specificity, and distance from the true community using Aitchison, Hellinger, and Bray-Curtis distance measures.

The results were striking. In the simulated communities, the choice between ASV and OTU methods had a statistically significant but comparatively small effect, whereas read processing, specifically whether the ITS region was extracted from merged paired-end reads or whether only forward reads were used, explained a larger share of the variation in community composition. On average, sensitivity was 12.7 percent higher for data that had undergone ITS extraction than for forward-read-only processing, and OTU methods were 15.9 percent more sensitive than ASV methods. Specificity, by contrast, varied little between treatments. The taxonomic level of analysis was consistently the single most important factor determining how closely a pipeline’s output matched the true community, with distances increasing at finer taxonomic resolutions.

Richness estimation revealed another clear pattern. Small sequencing libraries of 10,000 reads were insufficient to capture the full diversity of the simulated communities, underestimating species richness at nearly all diversity levels. Medium libraries performed better, and large libraries of 50,000 reads allowed both ASV and OTU pipelines with ITS extraction to produce accurate richness estimates, except in the highest-diversity communities containing multiple strains of the same species. The forward-read-only pipelines never managed to estimate richness accurately, and one of them, forward-read OTU clustering, actually overestimated richness, with the overestimate growing worse as library size increased. This finding directly contradicts fears that ASVs systematically inflate fungal diversity; in these simulations, ASVs tended to underestimate richness while OTUs tended to overestimate it, but the discrepancy largely vanished when ITS extraction and large library sizes were combined.

To confirm that their simulation results transferred to real sequencing data, the researchers reanalyzed a previously published 189-strain fungal mock community originally used to compare 260 bioinformatics pipelines. That earlier study had recommended using only forward reads with DADA2 ASVs and deemed ITS extraction unnecessary. Tosadori and Bosch reached very different conclusions, which they attribute to methodological differences: the original analysis required strict read-merging parameters with no mismatches allowed, used the older ITSx tool rather than ITSxpress, and relied on a custom database of Sanger sequences rather than the large reference databases standard in environmental microbiology. With more relaxed merging requirements, the joined reads retained far more information, and the benefit of ITS extraction became apparent. In the reanalysis, certain taxa were detected only in ITS-extracted samples while others appeared only in forward-read processing, and no orders or genera were specific to either ASVs or OTUs.

The practical implications are substantial. The authors recommend that researchers prioritize large library sizes, ideally around 50,000 reads per sample, which modern sequencing technology makes achievable with little extra effort. They further advise merging forward and reverse reads and performing ITS extraction unless there is a compelling reason not to, such as poor-quality reverse reads or species whose ITS regions are too long to merge, like chanterelles with ITS1 regions approaching 1,100 bases or the oomycete Plasmopara halstedii with an ITS2 region of roughly 2,200 bases. For pathogen surveillance or other applications requiring detection of specific taxa, analyzing data with both processing strategies may maximize the chances of correctly identifying the target organism. Notably, the team found no statistical benefit to fungal-specific modifications of DADA2 parameters that had been proposed to improve read retention, suggesting default settings remain appropriate.

The study does carry limitations the authors acknowledge. Their simulated communities were drawn only from fungi with fully sequenced genomes, a subset biased toward species that are easy to culture or of medical and commercial interest, and therefore not fully representative of natural fungal diversity. They also tested a limited, though realistic, set of processing options rather than exhaustively exploring every possible pipeline configuration. Standard short reads, moreover, lacked the resolution to distinguish strains of the same species, a task that requires full-length ITS sequences or alternative markers. Even so, the combination of simulated data with known ground truth and validation against a real mock community gives the findings unusual force in a field where method comparisons typically cannot determine which pipeline is closer to reality.

For a discipline that has invested considerable energy in the ASV-versus-OTU debate, the message is quietly liberating: for most fungal community studies, either method will yield broadly similar ecological conclusions, and researchers can choose based on their specific goals, favoring OTUs when sensitivity to particular taxa matters most and ASVs when minimizing false positives is the priority. The real leverage lies elsewhere, in sequencing deeply enough, joining paired-end reads, and carefully extracting the ITS barcode before analysis. As fungal metabarcoding continues to underpin research on soil health, plant pathology, climate change responses, and biodiversity monitoring, these evidence-based recommendations offer a clearer roadmap for study design than the field has previously possessed.

Subject of Research: Comparison of ASV and OTU bioinformatics pipelines for fungal ITS amplicon sequencing using simulated communities

Article Title: Decoding fungal communities: ITS extraction matters more than ASVs vs. OTUs

Article References: Tosadori, G., & Bosch, J. (2026). Decoding fungal communities: ITS extraction matters more than ASVs vs. OTUs. Web Ecology, 26(1), 47-59. https://doi.org/10.5194/we-26-47-2026

Image Credits: AI Generated

DOI: 10.5194/we-26-47-2026

Keywords: fungal ecology, metabarcoding, ITS region, ASVs, OTUs, amplicon sequencing, DADA2, bioinformatics pipelines, alpha diversity, ribosomal DNA, microbiome, study design

African animal trypanosomoses, caused by single-celled protozoan parasites of the genus Trypanosoma and transmitted exclusively through the bite of tsetse flies of the genus Glossina, remain one of the most formidable constraints on livestock productivity across sub-Saharan Africa. The disease devastates cattle herds, undermines plough-based agriculture, and imposes economic losses that ripple through rural communities already living at the edge of poverty. What makes the problem especially intractable is the ecology: tsetse flies feed on a wide range of vertebrate hosts, including wildlife that can carry trypanosomes without apparent illness, meaning that protected areas can act as reservoirs from which infection spills over into domestic animals and, occasionally, into people themselves.

Mozambique is a particularly instructive place to study this dynamic. The country harbours four main tsetse species, and pathogenic trypanosomes circulate widely in both wildlife and livestock. As human populations grow and agricultural activity intensifies around the edges of national parks, the interfaces where people, cattle, and wild animals meet have expanded, raising the risk of disease transmission in both directions. To understand exactly what is happening at these contact zones, the team concentrated its fieldwork on two contrasting landscapes: Maputo National Park in the south of the country and Gorongosa National Park in the centre, together with the agricultural buffer zones that surround each protected area.

The field effort was substantial. Using traps deployed inside the parks and in their buffer zones, the researchers collected 2,291 tsetse flies representing four species: Glossina austeni, Glossina brevipalpis, Glossina morsitans and Glossina pallidipes. Each specimen was identified using standard taxonomic keys, and a subsample of 200 flies was then selected for whole-body DNA extraction. From that genetic material, the team amplified and sequenced several molecular barcodes, targeting the internal transcribed spacer region to identify trypanosomes, mitochondrial markers to trace blood meal sources, and the 16S ribosomal RNA gene to profile the bacterial microbiome living inside each fly.

The trypanosome results were striking. Overall infection prevalence ranged from 65 to 90 percent depending on the fly species, an extraordinarily high parasite burden that underscores how intensively trypanosomes circulate in these landscapes. Seven distinct Trypanosoma species or subspecies were detected across the sampled flies. Trypanosoma brucei, the species group that includes the parasites responsible for human sleeping sickness, was the most abundant, found in 35 percent of flies, followed by Trypanosoma godfreyi at 20 percent. The team also identified Trypanosoma congolense Kilifi, Trypanosoma congolense Savannah, Trypanosoma simiae, Trypanosoma simiae Tsavo and Trypanosoma vivax, a roster that spans the full spectrum of parasites responsible for wasting diseases in cattle, pigs and other livestock.

Just as revealing was the blood meal analysis. By sequencing host DNA remaining in the flies’ guts, the researchers identified 17 different vertebrate host species, with wildlife predominating over domestic animals. Two hosts stood out at the top of the list: warthogs accounted for 26 percent of identified blood meals, and humans accounted for 25 percent. That human blood appears so frequently in the diet of these flies is a sobering finding, because it demonstrates that the ecological circuit connecting wild reservoirs, vectors and people is not hypothetical but an everyday occurrence. Cattle and other domestic animals also featured among the identified sources, confirming that livestock are woven into the same transmission network rather than standing apart from it.

The microbiome profiling added a third layer to the picture. Inside the flies, the dominant bacterial symbionts were Wigglesworthia and Wolbachia, two genera with well-known roles in tsetse biology. Wigglesworthia is an obligate mutualist housed in specialised bacteriomes that supplies nutrients the fly cannot obtain from its blood-only diet, and it has been implicated in vector competence and fertility. Wolbachia, by contrast, is a notorious reproductive manipulator that can bias host sex ratios and is being explored worldwide as a tool for controlling insect-borne diseases. Mapping which bacteria coexist with which parasites in which fly species provides raw material for future biocontrol strategies that might exploit these internal partnerships.

Perhaps the most important analytical finding concerns what actually shapes this entire system. When the researchers modelled the relationships among trypanosome diversity, host selection and microbiome composition, they found that the species of tsetse fly was the dominant explanatory factor, while location, meaning the difference between the parks and their surrounding buffer zones, played only a secondary role. In other words, the biology of the vector itself, rather than the immediate landscape it inhabits, is the strongest organising force in the transmission cycle. A constrained ordination analysis of blood meal variation showed that tsetse species together explained 96 percent of the total variance in community composition, a statistically significant association that highlights how tightly each fly species is bound to its own host preferences and parasite suite.

This species-level structure has practical consequences. Different Glossina species occupy different ecological niches, prefer different hosts, and harbour different combinations of trypanosomes, so a single blanket intervention is unlikely to succeed everywhere. Control programmes in the Gorongosa region, where G. morsitans and G. pallidipes dominate, may need different tactics from those around Maputo, where G. austeni and G. brevipalpis prevail. The finding that wildlife, particularly warthogs, sustain so many blood meals suggests that reservoir management and vector control must be designed together, and the prominence of humans in the blood meal record argues for surveillance of human African trypanosomosis even in areas where it is not currently considered a major clinical problem.

The study’s authors frame their results within a One Health perspective, the recognition that human, animal and environmental health are inseparable. By documenting the full diversity of vectors, parasites, hosts and microbes at the conservation–agriculture interface, the research provides the evidence base needed to design targeted, locally appropriate control strategies rather than generic ones. Funded by the European Union’s Horizon 2020 programme through the COMBAT project, which aims to control and progressively minimise the burden of animal trypanosomosis, the work demonstrates how molecular tools can transform our understanding of neglected tropical disease systems. As agricultural frontiers continue to press against Africa’s protected areas, studies like this one make clear that the fate of livestock, wildlife and people will be decided in the shared landscapes between them, one tsetse bite at a time.

Subject of Research: Tsetse fly-borne trypanosome transmission, blood meal hosts and microbiome diversity at wildlife–livestock–human interfaces in Mozambique

Article Title: Tsetse flies, trypanosomes, hosts and microbiome diversity at agricultural–conservation interfaces in Mozambique

Article References: Brito, D. R. A., Mulandane, F. C., Cossa, N. V., Mucache, H. N., Moo-Millan, J. I., Manzanilla, V., Boulangé, A., Waleckx, E., & Neves, L. (2026). Tsetse flies, trypanosomes, hosts and microbiome diversity at agricultural–conservation interfaces in Mozambique. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07660-2

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07660-2

Keywords: tsetse flies, trypanosomes, African animal trypanosomosis, Mozambique, Glossina, blood meals, microbiome, Wigglesworthia, Wolbachia, One Health, wildlife reservoirs, metabarcoding

Gastrointestinal nematodes are among the most economically consequential pathogens of grazing livestock worldwide. In cattle, species such as Haemonchus placei, a blood-feeding abomasal worm, and various Cooperia species that damage the small intestine cause weight loss, reduced milk yield, poor fertility and, in heavy infections, death. The global cost is measured in billions of dollars annually, and the burden falls disproportionately on smallholder and pastoralist communities across sub-Saharan Africa, where livestock are both income and insurance. Yet the epidemiology of these worms in Africa remains strikingly under-characterised. Most surveillance still depends on faecal egg counts, which estimate the number of eggs shed per gram of faeces, and on the microscopic identification of third-stage larvae cultured from faecal samples. Both approaches have fundamental limitations that the new study set out to address.

Faecal egg counts cannot distinguish between species, because the eggs of many strongylid nematodes are morphologically near-identical. Larval culture and identification improve on this, but the technique is laborious, requires skilled parasitologists, takes one to two weeks, and is subject to culture biases that can distort the apparent composition of an infection. Different species develop and survive in culture at different rates, meaning the larval population recovered in a petri dish may not faithfully represent the worm population inside the animal. In tropical settings, where ambient temperatures accelerate larval development and where laboratory infrastructure may be limited, these problems are compounded. The result is a sparse and potentially skewed picture of nematode diversity across much of the continent, precisely where that diversity matters most for designing effective control.

The nemabiome approach sidesteps these constraints by reading the parasites’ DNA directly. The method targets the internal transcribed spacer-2 region, or ITS-2, a stretch of ribosomal DNA that varies sufficiently between species to serve as a barcode. Using primers that amplify this region from any Clade V nematode, the strongylid group that includes the major livestock worms, researchers can sequence thousands of amplicons in parallel from a single sample. Bioinformatic pipelines then sort the resulting reads into species-level operational taxonomic units and estimate their relative abundances. Because the technique works on any life stage containing usable DNA, it can in principle be applied to eggs in faeces, to first-stage larvae hatched from those eggs, or to third-stage larvae recovered from culture, allowing researchers to compare what each sample type reveals.

That is exactly what the team did at Ol Pejeta, a conservancy where livestock grazing is deliberately integrated with wildlife conservation, including populations of elephants, rhinos and large predators. The study focused on adult cows and their calves, sampling faeces, first-stage larvae and cultured third-stage larvae from individual animals. The nemabiome analysis confirmed infections with eight species: Cooperia curticei, Cooperia pectinata, Cooperia punctata, Haemonchus placei, Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Several of these findings are notable in themselves. Haemonchus contortus, classically a parasite of sheep and goats, was detected in cattle, raising questions about cross-species transmission at the interface. Teladorsagia circumcincta, another small-ruminant worm, likewise appeared in the cattle nemabiome, a signal that would have been easy to miss with morphology-based methods alone.

Perhaps the most striking quantitative finding concerns coinfection. Mixed-species infections accounted for 77.4 percent of all infections detected in the study animals. In other words, the typical infected cow at Ol Pejeta was not carrying a single worm species but a community of them, with the composition varying from animal to animal. This matters because nematode species differ in their pathogenicity, their seasonal dynamics and, critically, their response to anthelmintic drugs. A treatment regimen informed only by faecal egg counts treats the infection as an undifferentiated mass; a regimen informed by metabarcoding can recognise, for example, that a Haemonchus-dominated infection may demand different management than one dominated by Trichostrongylus. The study’s authors argue that uncovering this diversity opens new opportunities to investigate interactions between worm species within the host, including competition, facilitation and their consequences for disease severity and drug efficacy.

Age emerged as a second axis of variation. Calves carried higher infection rates than their dams and harboured more diverse nematode communities. This pattern is consistent with the immunology of helminth infection: adult cattle gradually acquire partial immunity through repeated exposure, which suppresses worm establishment and fecundity, whereas young animals, still immunologically naive, accumulate infections more freely. From a control perspective, the finding reinforces the importance of monitoring calves specifically, since they are both the most heavily parasitized cohort and the one in which subclinical damage to gut and abomasum can permanently compromise growth. It also suggests that dam–calf pairs provide a natural experiment for studying the development of nemabiome diversity over time, a direction the authors see as promising for future work.

Equally consequential is the study’s practical demonstration of which sample types can carry the analysis. Faecal samples and first-stage larvae both proved reliable sources of nematode DNA, yielding species compositions consistent with the gold-standard cultured third-stage larvae. This is a significant result for tropical field settings. Third-stage larval culture requires incubation, careful moisture and temperature management, and weeks of waiting; faeces require a bag and a courier. If routine surveillance can be run on faecal or first-stage larval DNA, the barrier to implementing molecular monitoring in African veterinary services drops dramatically. The authors position this as offering a practical alternative to labour-intensive L3 culturing, and the implication for national control programmes is clear: species-level surveillance could become as routine as the egg counts it would complement.

The wildlife–livestock interface setting adds a further layer of significance. Ol Pejeta’s cattle graze land shared with wild ungulates, and several of the nematode species detected are known to infect multiple host species. Haemonchus contortus, for instance, circulates in wild ruminants as well as small livestock, and Trichostrongylus species have broad host ranges. Molecular barcoding of livestock parasites therefore offers a window into transmission dynamics that span the domestic–wild boundary, something morphology-based diagnostics have never been able to resolve at scale. The authors argue that in such complex ecosystems, nemabiome approaches provide strong potential for unbiased disease surveillance, capturing the full parasite community rather than only the species a technician expects to find. As land-use change brings livestock and wildlife into ever closer contact across East Africa, tools that can track parasite flow between them will become increasingly valuable for both conservation and animal health.

The study also serves as a proof of concept for building African nemabiome reference data. Metabarcoding is only as good as its reference database; reads can be assigned to species only when that species’ ITS-2 sequence has been catalogued. By generating and validating species assignments in a Kenyan herd, the team has contributed to the baseline needed for wider deployment across the continent. Funded in part by the Bill & Melinda Gates Foundation and UK aid through the Centre for Tropical Livestock Genetics and Health, the work reflects a growing investment in molecular epidemiology for African livestock systems. The immediate next steps, the authors suggest, include exploiting the revealed diversity to study inter-species interactions within hosts and to track how nematode communities shift with season, treatment and grazing management. For a class of parasites that has long been counted but rarely identified, the era of seeing the whole community has begun.

Subject of Research: Gastrointestinal nematode diversity and coinfection in Kenyan cattle at a wildlife–livestock interface, assessed by nemabiome metabarcoding

Article Title: Gastrointestinal nematode diversity in Kenyan cattle at wildlife–livestock interfaces: a deep-amplicon sequencing approach

Article References: Yalcindag, E., Karani, B. E., Vasoya, D., van Bunnik, B. A. D., Freeman, E. J., Ngulu, S., van Aardt, R., Toye, P., Sargison, N., Morrison, L. J., & Bronsvoort, B. M. D. C. (2026). Gastrointestinal nematode diversity in Kenyan cattle at wildlife–livestock interfaces: a deep-amplicon sequencing approach. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07657-x

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07657-x

Keywords: gastrointestinal nematodes, nemabiome, metabarcoding, Kenya, cattle, wildlife–livestock interface, Haemonchus, Cooperia, coinfection, parasite surveillance, ITS-2 sequencing, livestock health

The research, led by Anthony M. Bonacolta and Javier del Campo of the University of Miami’s Rosenstiel School of Marine, Atmospheric, and Earth Science, together with colleagues in Miami and Barcelona, focused on the mountainous star coral, Orbicella faveolata, a major reef-building species in the Caribbean and Gulf of Mexico. Under controlled heat-stress conditions in the laboratory, the team tracked the coral as it progressed from a healthy state through mid-bleaching stages to full bleaching. What makes the study unusual is its resolution: rather than averaging gene activity across an entire coral fragment, the researchers sequenced RNA from individual cells, allowing them to see which cell types were responding, when, and in what direction.

The technical foundation of the work is single-cell RNA sequencing, or scRNA-seq, a method that captures the transcriptome, the complete set of active genes, of thousands of individual cells simultaneously. The team combined this with marker gene metabarcoding targeting three genetic regions at once: the 16S rRNA gene for bacteria and archaea, the 18S rRNA gene for microbial eukaryotes, and the ITS2 region to distinguish types of algal symbionts. Photochemical measurements of the algae’s photosynthetic performance rounded out the picture. Together, these approaches produced what the authors describe as a granular view of the microbial ecology of symbiotic breakdown, treating the coral not as a single organism but as a holobiont, an integrated ecosystem of host cells, algae, bacteria, and protists.

One of the most striking findings concerns the two co-dominant algal symbionts living within the same coral colonies. Orbicella faveolata hosts both Durusdinium and Breviolum, two genera within the family Symbiodiniaceae that provide the coral with most of its energy through photosynthesis. When heat stress arrived, the two symbionts did not respond identically. Their transcriptomic diverged particularly sharply in transcripts involved in nitrogen cycling, a metabolic arena that sits at the heart of the symbiotic bargain. The coral supplies nitrogen-poor compounds to its algae in exchange for carbon-rich photosynthate, and the new data suggest that the way each symbiont genus handles nitrogen under stress may shape how the partnership unravels.

The host side of the story proved equally revealing. The researchers identified distinct coral cell types, including gastrodermal cells, the digestive cells that physically house the algal symbionts, and gland cells known as mucocytes, which produce mucus. In the gastrodermal cells, the team observed something unexpected: the suppression of host heat-stress genes differed depending on which symbiont a given cell was hosting. In other words, the identity of the algal tenant appeared to influence how the coral cell itself expressed its stress response, a symbiont-specific effect visible at the level of individual host cells. This finding hints that the two partners in each hosting cell are chemically conversing in ways that standard bulk measurements would completely miss.

The mucocytes delivered perhaps the most dramatic cellular storyline. A subpopulation of these mucus-producing gland cells showed a distinct increase during heat stress, suggesting that they are not passive bystanders in bleaching but active participants in the response. Mucus production is a well-known feature of stressed corals, often interpreted as a protective or sloughing mechanism, but the single-cell data now place mucocytes squarely within the cellular choreography of the bleaching process. The finding gives researchers a specific cell type to watch, and potentially to target, when trying to understand why some colonies survive thermal anomalies that kill others.

Beyond the coral and its algae, the study broke new ground by documenting, for the first time, the concurrent shifts in both the prokaryotic and microeukaryotic microbiomes during experimental heat stress. The prokaryotic community told a coherent metabolic story. Early in the heat-stress response, suspected nitrifying bacteria, microbes that convert ammonia into nitrate, decreased, while suspected denitrifiers and nitrate reducers increased. The net effect is a rise in nitrogen availability within the coral’s internal environment. This matters because nitrogen availability is theorized to regulate the size of the Symbiodiniaceae population: when nitrogen is scarce, the coral can keep its algal partners in check. More nitrogen may decouple that regulation, allowing algal populations to expand or behave in ways the host can no longer control.

The consequences rippled outward through the rest of the holobiont. The higher nitrogen availability coincided with blooms of other microbes, including chlorophytes, a group of green algae, diatoms, and labyrinthulids, slime-net-producing protists that are often overlooked in coral studies. These opportunistic eukaryotic microbes may contribute to the negative feedback loops that characterize coral bleaching, in which the initial stress response creates conditions that accelerate further dysfunction. The picture that emerges is not a simple two-partner divorce between coral and alga but a cascading ecological rearrangement of an entire microscopic community, with nutrient cycling at its center.

The study’s conclusions position it as a starting point rather than an endpoint. By pairing scRNA-seq with the latest metabarcoding methodologies, the authors provide what they call a comprehensive view of the coral holobiont, one in which all members, from gastrodermal cells to denitrifying bacteria to labyrinthulid protists, are considered interconnected and important to the health of the whole. The cell-type-specific responses they documented, particularly within mucocytes and gastrodermal cells, along with the symbiont-specific suppression of heat-stress transcripts, offer concrete mechanistic threads for future work on symbiosis breakdown, coral mortality, and ultimately reef decline.

For a field racing against warming oceans, the significance of this cellular atlas is hard to overstate. Bleaching events are becoming more frequent and severe, and predictions of reef futures depend on understanding why some corals bleach and die while others endure. By revealing that the answer may lie partly in which symbiont genus a cell hosts, how its mucocytes respond, and whether its bacterial community tips from nitrification toward denitrification, the study converts a planetary catastrophe into a set of testable, cell-level hypotheses. The mountainous star coral that yielded these insights now serves as a reference point for the single-cell ecology of the holobiont, and the methods deployed here, from STARsolo-based RNA mapping to ANCOM-BC differential abundance analysis and cross-species cell-type matching against existing coral atlases, give other laboratories a template for extending this work to more species, more symbiont combinations, and the real thermal landscapes of dying and surviving reefs.

Subject of Research: Single-cell analysis of coral-algal symbiosis breakdown and holobiont microbiome shifts during heat stress

Article Title: Single-cell ecology of coral-algal symbiosis breakdown

Article References: Single-cell ecology of coral-algal symbiosis breakdown. (n.d.). https://doi.org/10.1186/s40168-026-02506-6

Image Credits: AI Generated

DOI: 10.1186/s40168-026-02506-6

Keywords: coral bleaching, single-cell transcriptomics, Symbiodiniaceae, holobiont, microbiome, Orbicella faveolata, Durusdinium, Breviolum, nitrogen cycling, metabarcoding, mucocytes, gastrodermal cells

The research, published in the journal Microbial Ecology, was carried out by a team from the University of Padova and the National Biodiversity Future Centre in Italy. Led by Irene Gregori and corresponding author Alessandro Vezzi, the researchers sampled sediments from two salt marshes in the Venice Lagoon, a productive microtidal coastal ecosystem in the northern Adriatic. Rather than tracking the marshes across weeks or seasons, the team focused on a much finer timescale: a single summer diel cycle, with sampling at four carefully chosen moments: sunset, night, dawn and solar noon. This design allowed them to capture the most extreme short-term physicochemical oscillations that intertidal ecosystems experience, driven by the interplay of sunlight, tides and biological activity.

The physicochemical measurements confirmed just how dynamic these environments are. Temperature and dissolved oxygen concentrations showed pronounced oscillations over the sampled cycle, with oxygen levels in particular responding to the balance between photosynthetic production during daylight hours and continuous respiratory consumption around the clock. Such fluctuations are typical of productive intertidal systems, where mats of microalgae and aquatic plants flood the water with oxygen by day and leave it depleted by night. For organisms living in the sediment, these swings represent a genuine physiological challenge, because oxygen availability dictates which metabolic pathways are energetically feasible at any given moment.

To characterise the biological players, the team employed two complementary DNA-based approaches. Prokaryotic communities, encompassing bacteria and archaea, were profiled using metabarcoding of the 16S ribosomal RNA gene, a standard molecular fingerprinting technique that reveals which microbial taxa are present and in what relative abundances. Eukaryotic organisms, ranging from microscopic algae to fungi and protists, were similarly catalogued using the 18S rRNA gene. But the researchers went a step further than simple taxonomic inventories: they also applied metagenomics, sequencing the collective genomes of the prokaryotic communities to assess their functional potential, in other words, which genes and metabolic pathways the communities carried and could, in principle, deploy.

The results were striking for what they did not show. Despite the pronounced swings in temperature and dissolved oxygen, both the prokaryotic and eukaryotic communities displayed only minor shifts in taxonomic composition across the four sampling points of the diel cycle. The same microbial lineages dominated the sediments at sunset as at dawn, and the relative abundances of most taxa remained essentially unchanged from night to solar noon. In many aquatic ecosystems, particularly the open ocean, researchers have documented clear diel rhythms in microbial communities, with different taxa rising and falling in abundance as light and nutrient conditions change. The Venice Lagoon salt marsh sediments, by contrast, appear to host assemblages that simply do not need to restructure themselves on this timescale.

The metagenomic data help explain why. The prokaryotic communities proved to be functionally redundant and metabolically versatile, meaning that multiple different taxa carried out the same key processes and that individual organisms possessed a broad repertoire of metabolic capabilities. The communities were dominated by taxa involved in organic-matter degradation, the cycling of sulphur compounds, denitrification, which converts bioavailable nitrogen into gaseous forms, and sulphur-oxidising chemolithoautotrophic carbon fixation, a process in which microbes use chemical energy from oxidising sulphur compounds, rather than sunlight, to fix carbon dioxide into biomass. This functional breadth means that whatever the prevailing oxygen and temperature conditions at a given hour, some members of the community are equipped to keep the essential biogeochemical machinery running.

The authors conclude that modulating their activities, rather than undergoing compositional restructuring, is likely how these communities sustain key ecological processes relevant to carbon and nutrient cycling over the diel timescale. In practical terms, a sulphate-reducing bacterium that dominates the community at midnight, when oxygen has been exhausted, does not need to be replaced by a different species at noon; it can simply slow down or speed up its metabolic output as conditions permit. Similarly, chemolithoautotrophs that fix carbon using sulphur-derived energy can ramp up their activity when their substrates become available, without any change in who is present. Activity-level regulation is a faster and energetically cheaper strategy than community turnover, and the study suggests it is the dominant mode of short-term response in these sediments.

The findings carry broader significance for the conservation and restoration of salt marshes, which are globally threatened by anthropogenic pressures including land reclamation, pollution, nutrient loading and sea-level rise. Because restoration efforts are increasingly needed to recover the carbon sequestration capacity, water quality regulation and coastal protection that these habitats provide, understanding the stability and resilience of their microbial engines is essential. If sediment microbial communities are functionally robust in the face of strong short-term physicochemical fluctuations, this suggests a degree of intrinsic resilience that could help restored marshes establish functioning biogeochemical cycles relatively quickly. At the same time, the central role of sulphur cycling, denitrification and organic-matter degradation in these communities underscores how tightly salt marsh ecosystem services are coupled to microbial metabolism, and how disturbances that alter sediment chemistry could ripple through the entire system.

The study also highlights the value of combining fine-scale temporal sampling with functional genomics. Taxonomic metabarcoding alone might have led researchers to conclude that diel dynamics are unimportant in salt marsh sediments, but the metagenomic perspective reveals the hidden versatility that makes that stability possible. As sequencing costs continue to fall, similar integrated approaches are likely to be applied to other intertidal and coastal ecosystems, from mangrove sediments to seagrass meadows, where the same questions about short-term microbial dynamics and ecosystem function remain open. For now, the Venice Lagoon results provide a clear and somewhat reassuring message: beneath the mud, the microbial workforce keeps its composition steady through night and day, adjusting its activity to whatever the marsh throws at it, and in doing so quietly sustaining the processes on which these threatened coastal habitats depend.

Subject of Research: Diel stability of sediment microbial communities in Venice Lagoon salt marshes

Article Title: Night and day in the Salt Marshes: Sediment Microbial Communities Remain Stable Over a Summer Diel Cycle

Article References: Gregori, I., Mohamed, F., Barausse, A., Frizzo, R., Archetti, L., Martino, F., Zane, L., & Vezzi, A. (2026). Night and day in the Salt Marshes: Sediment Microbial Communities Remain Stable Over a Summer Diel Cycle. Microbial Ecology. https://doi.org/10.1007/s00248-026-02890-4

Image Credits: AI Generated

DOI: 10.1007/s00248-026-02890-4

Keywords: salt marshes, microbial ecology, metabarcoding, metagenomics, diel cycle, Venice Lagoon, biogeochemistry, sulphur cycling, denitrification, carbon sequestration, coastal ecosystems, prokaryotes

The research team, led by Hee Jin Chung of the Mammalian Ecology and Conservation Unit at UC Davis’s Veterinary Genetics Laboratory, recruited owners of indoor–outdoor cats in Davis and Sacramento, California. Participants were given simple kits containing ethanol-filled tubes, nitrile gloves, and a printed log, and were asked to collect five of their cat’s fecal samples over several weeks, along with a sample of whatever food the cat was being fed. Between November 8 and December 21, 2023, the team received 38 scats from eight neutered house cats—three males and five females—whose samples were, for anonymity, labeled Cat A through Cat H. Most samples came straight from litter boxes and were collected within hours of defecation, making them unusually fresh for molecular work.

The technical core of the study was DNA metabarcoding, a sequencing approach that identifies the mixture of DNA left behind in feces. The researchers amplified two mitochondrial markers: the 12S rRNA gene to detect vertebrate prey and the 16S rRNA gene to detect invertebrates. Because cat DNA overwhelmingly dominates cat feces, the team designed a custom blocking oligonucleotide—a short synthetic molecule carrying a three-carbon spacer—that binds to domestic cat sequences and stalls their amplification, allowing dietary DNA to be recovered more easily. Sequencing was performed on an Illumina platform, and the resulting reads were processed with cutadapt and DADA2, which identify distinct amplicon sequence variants, before taxa were assigned using BLAST searches against custom and NCBI reference libraries. Samples below strict read-count and identity thresholds were discarded to guard against contamination.

The results were striking, and somewhat humbling for anyone who assumes their cat is quietly devastating the neighborhood. Of 32 analyzable scat samples, roughly a third contained no vertebrate DNA at all except that of the cat itself. Most of the remainder revealed only anthropogenic food items—chicken, pig, turkey, and fish from the drum, hake, and herring families—matching the provisioned pet foods owners submitted for comparison. In total, only a single scat, produced by Cat H, contained wild vertebrate prey: the California vole, Microtus californicus, a common rodent in the region. The cat that produced it had not been seen with any prey by its owner in the preceding 24 hours.

Even more revealing were the three cases where owners did witness their cats carrying prey—a live house mouse caught indoors, a small unidentified rodent brought to the door, and a lizard caught in front of the home. In every instance the prey was alive and intact, and in every instance the corresponding scat, produced within 24 hours, contained no wild prey DNA whatsoever. The findings suggest that domestic cats frequently hunt without consuming what they catch, consistent with earlier camera-collar studies showing that satiated cats hunt for enrichment, practice, or caching rather than nutrition. They also imply that significant prey consumption is needed before prey DNA becomes detectable in feces, meaning molecular scat analysis and owner reports capture different facets of the same hunting behavior—and should be used together.

The comparison between provisioned food and scat also yielded a methodological surprise. Chicken and pig DNA appeared in all four collected food samples, as expected, but recovery of those ingredients in the corresponding scats varied widely. Chicken, present in nearly every relevant food, showed up in 14 of 15 expected scat samples, making it the most digestible and detectable ingredient. By contrast, the two prescription diets—a hydrolyzed-protein formula and a kidney-care wet food—showed the poorest post-digestion recovery, likely because heavy industrial processing degrades DNA before the food even reaches the bowl. Notably, several scats from cats eating animal-based diets contained no animal DNA other than cat DNA, a finding with implications beyond cats: urban wildlife studies routinely identify pet food as an anthropogenic food source in carnivore scats, and this study suggests such DNA may often be too degraded to detect.

The team also tested a cost-saving shortcut that could matter for future studies: pooling. Because DNA extraction and sequencing are expensive, they homogenized subsamples of three to five scats from each cat into a single pooled sample, then compared pooled results against individually extracted samples. The answer was mixed. Common items such as chicken were recovered reliably regardless of pooling, and for two cats the two approaches matched perfectly. But the study’s only wild prey detection—the California vole—appeared only in the individually extracted sample and vanished in the pooled version. When the target of a study is a rare or rarely consumed prey item, the authors conclude, pooling samples before extraction can erase exactly the signal researchers are looking for.

The invertebrate results illustrate another caution. The 16S data were compromised by heavy contamination in negative controls and poor amplification, and although Orthoptera, flies, true bugs, jumping spiders, and noctuid moths were detected in some scats, the team excluded the invertebrate dataset from formal analysis. Primer bias is a further limitation: the 12S primers used here perform poorly on reptiles and amphibians, so the absence of lizard DNA in the scats—despite one owner reporting a caught lizard—may reflect molecular bias rather than true absence. The authors recommend that future studies pair molecular methods with morphological analysis of scat contents to avoid missing entire taxonomic groups.

Despite its small sample size and short duration—cats were sampled for an average of about 12 days—the study demonstrates that owned cats, paired with their willing owners, can serve as a viable study system for predator diet research. Citizen-science sample collection slashes the cost and effort of fieldwork, while owner logs add behavioral context that scat alone cannot provide. And because neither sterilization nor regular feeding reliably suppresses hunting, even well-fed, neutered cats remain relevant to wildlife conservation, particularly where vulnerable prey such as the endangered Amargosa vole, a California vole subspecies, persist. The broader lesson is that a single tool will never fully capture the ecology of a predator that hunts both out of hunger and out of instinct. DNA metabarcoding catches the meals owners never see; owner questionnaires catch the trophies cats bring home uneaten. Used together, they offer the clearest picture yet of what the world’s most popular pet is really doing to the wildlife in its own backyard.

Subject of Research: DNA metabarcoding of owner-collected scat samples to assess the diet and wild prey consumption of owned domestic cats.

Article Title: Using DNA Metabarcoding and Citizen Science to Assess the Diet of Owned Domestic Cats

Article References: Chung, H. J., Vanderzwan, S. L., & Sacks, B. N. (2026). Using DNA Metabarcoding and Citizen Science to Assess the Diet of Owned Domestic Cats. Ecology and Evolution, 16(9), Article e74404. https://doi.org/10.1002/ece3.74404

Image Credits: AI Generated

DOI: 10.1002/ece3.74404

Keywords: domestic cat, DNA metabarcoding, citizen science, diet analysis, wildlife conservation, California vole, pet food, hunting behavior, scat samples, owned cats, Metabarcoding, Citizen