The study, led by Lorenzo Zacchetti of the National Research Council’s Institute for Biological Resources and Marine Biotechnologies in Ancona, together with colleagues from the University of Bologna, the National Biodiversity Future Center, the CNR Institute of Research on Terrestrial Ecosystems and Liverpool John Moores University, introduces what the authors call the Gut-Isotope-DNA, or GID, approach. According to the researchers, this is the first time such an integrated framework has been applied to a crustacean. The method weaves together three techniques that each carry different strengths and blind spots: gut content analysis, which identifies prey visually but only captures recently ingested and recognisable items; stable isotope analysis, which integrates diet over weeks to months but resolves prey only at broad categories; and DNA metabarcoding, which can detect even soft, digested or unrecognisable prey from trace genetic material.

The fieldwork took place in one of the most intriguing features of the Adriatic basin: the Pomo Pits, two deep depressions in the middle of the sea that host a legally protected Fishery Restricted Area. Specimens of Norway lobster were collected both inside and outside this protected zone during two separate surveys, in 2019 and 2023, giving the team a rare opportunity to test whether diet and gut microbiome differ between a heavily fished area and a refuge, and whether these patterns shift between years separated by a four-year gap. The samples were gathered during scientific trawl surveys funded by the Italian Ministry of Agricultural, Food, and Forestry Policies in 2019 and by the European H2020 NAUTILOS project in 2023, which was designed to validate new low-cost ocean observation instruments on fishing vessels.

When the researchers opened up the lobsters’ guts and examined the contents under the microscope, one prey species towered above all others: the squat lobster Iridonida speciosa, a small relative of the Norway lobster that shares the same muddy habitat. This visual finding was not a fluke of preservation. Stable isotope mixing models, which use the ratios of carbon and nitrogen isotopes in the predator’s tissues to estimate the proportions of different food sources, independently pointed to the same conclusion. Together, the two methods painted a picture of an animal whose diet is anchored on a single abundant resource, at least in the parts of its menu that are large and durable enough to survive digestion intact.

But the DNA metabarcoding results told a richer and more nuanced story. By amplifying and sequencing genetic markers from the gut contents, the team detected a much wider array of prey than visual inspection had revealed, including soft-bodied organisms and fragments that would never survive to be identified under a dissection microscope. The molecular data exposed the Norway lobster as a genuine mixed scavenger-predator, opportunistically consuming both live prey and carrion across the seafloor. Intriguingly, the dietary diversity recorded through metabarcoding was higher in 2019 than in 2023, and finfish contributed more to the diet in the earlier year. This temporal pattern held both inside and outside the Pomo Pits, suggesting that shifts in food availability or feeding behaviour between the two survey years rippled through the population regardless of protection status.

The most striking twist came from the gut microbiome. Sequencing the bacterial communities living inside the lobsters’ digestive tracts revealed a microbial world dominated by three families: Vibrionaceae, a group of marine bacteria that includes both harmless associates and notable pathogens; Erysipelotrichaceae; and Mycoplasmataceae, cell-wall-less bacteria frequently found in the guts of crustaceans and other animals. These microbial residents were not passive bystanders. Their composition tracked the lobsters’ trophic patterns closely, with specimens caught outside the Pomo Pits carrying significantly more diverse gut microbial communities than those from inside the protected area.

What surprised the team most was the discriminating power of the microbiome itself. When the researchers asked which data stream best separated lobsters by sampling area and by year, the gut microbial communities outperformed the strictly dietary analyses. In other words, the invisible ecosystem within the gut acted as a sensitive recorder of where and when an animal lived and fed, sometimes more sharply than the food remains it carried. This finding raises the tantalising prospect that gut microbiomes could serve as biological indicators of habitat use and environmental conditions in commercially exploited species, complementing conventional tools such as stomach content analysis and population genetics.

The study also carries a methodological caution that the authors are careful to underline. While dietary composition and gut microbiome structure in the Norway lobster appeared broadly consistent with one another, each of the three techniques in the GID approach homes in on a different biological phenomenon. Gut content analysis captures a snapshot of recent, identifiable meals. Stable isotopes integrate assimilated diet over longer timescales but blur fine taxonomic detail. DNA metabarcoding maximises taxonomic resolution and detects trace prey but can be influenced by digestion rates, primer bias and the differential survival of DNA across prey types. Integrating evidence from all three therefore requires tailored interpretations, the researchers argue, and careful consideration of what each line of evidence can and cannot support, particularly when conclusions feed into fisheries management decisions.

That management dimension is far from academic. Nephrops norvegicus, known on dinner plates as scampi or langoustine, is one of the most economically important shellfish resources in European waters, and its stocks are assessed and regulated through effort controls, minimum landing sizes and area closures such as the Pomo Pits Fishery Restricted Area. Understanding what the species eats, where it forages and how its feeding ecology responds to protection and to environmental change is central to building sustainable management plans. If the lobster’s diet shifts between years, as the 2019 to 2023 comparison suggests, then trophic models underpinning stock assessments may need to account for that variability rather than assuming a static food web.

The GID framework, its developers suggest, could now be extended beyond a single species. Any crustacean, fish or benthic invertebrate whose diet is hard to observe directly could be profiled with the same triple lens, and the microbiome component adds a layer of ecological information that traditional diet studies simply do not capture. As sequencing costs continue to fall and reference databases of prey DNA grow, the approach may become a routine instrument in the marine ecologist’s toolkit. For now, the humble Norway lobster has become the first crustacean to have its meals, its chemistry and its inner microbial world read together, and the reading suggests an animal far more flexible, opportunistic and microbially distinctive than its muddy burrow home would ever let on.

Subject of Research: Dietary ecology and gut microbiome of the Norway lobster assessed through an integrated gut content, stable isotope and DNA metabarcoding approach

Article Title: Integrating Dietary Characterisation and Gut Microbiome Analysis in Norway Lobster (Nephrops norvegicus) Through Gut-Isotope-DNA (GID) Approach

Article References: Zacchetti, L., Basili, M., Domenichetti, F., Quero, G. M., Lauteri, M., Mariani, S., Manini, E., & Martinelli, M. (2026). Integrating Dietary Characterisation and Gut Microbiome Analysis in Norway Lobster (Nephrops norvegicus) Through Gut-Isotope-DNA (GID) Approach. Microbial Ecology. https://doi.org/10.1007/s00248-026-02902-3

Image Credits: AI Generated

DOI: 10.1007/s00248-026-02902-3

Keywords: Norway lobster, Nephrops norvegicus, gut microbiome, DNA metabarcoding, stable isotopes, trophic ecology, Adriatic Sea, Pomo Pits, fisheries management, Vibrionaceae, food web, marine ecology

The research, led by Marino Korlević of the Ruđer Bošković Institute in Rovinj, Croatia, together with colleagues from several Croatian marine research institutions, set out to answer a deceptively simple question: which microbes genuinely live on mussel gills, and which are merely passing through? This distinction matters because resident taxa may perform functions that benefit the host, such as contributing to nutrition or defense, while transient taxa simply reflect what the animal has recently filtered from the water column. Separating the two requires sampling that spans both space and time, which is precisely what the team undertook.

The researchers sampled mussels at two locations on the eastern coast of the Adriatic Sea, Lim Bay on the Istrian peninsula and Mali Ston Bay in Dalmatia, two of Croatia’s most important shellfish farming areas. Crucially, they did not stop at the mussels themselves. At the same time and at the same locations, they also characterized the microbial communities in the surrounding seawater and in the sediment. This three-way comparison is what gives the study its analytical power: if a microbe found on the gills is also abundant in seawater, it may simply be a hitchhiker; if it appears on the gills regardless of its abundance in the environment, it is more likely a true associate of the host.

To characterize the communities, the team used Illumina MiSeq sequencing of the V4 region of the 16S rRNA gene, the standard workhorse of modern microbiome research. This approach amplifies and reads a short, highly conserved stretch of bacterial DNA that allows researchers to identify which taxa are present and in what relative proportions. Sequences were grouped into operational taxonomic units, or OTUs, providing a fine-grained picture of community structure that could be compared across individuals, sites, and months.

The first major finding was that the gill microbiome is genuinely distinct. When the researchers compared the structure of the gill communities, measured at the OTU level, with the microbial communities of the surrounding seawater and sediment, the gills stood apart from both. This is not a trivial result. Mussels pump enormous volumes of water across their gills every day, and if the gill community were simply a snapshot of the planktonic bacteria in that water, the two communities would look statistically indistinguishable. The fact that they do not suggests that the gill environment actively selects for particular microbes, whether through the gill’s epithelial surfaces, its mucus chemistry, the immune defenses of the host, or the microconditions of oxygen and nutrients that prevail there.

Equally striking was what the comparison between the two bays showed, or rather did not show. The structure of the gill microbiome did not differ significantly between Lim Bay and Mali Ston Bay, despite the geographic separation and the environmental differences between the sites. In other words, whatever forces shape the mussel gill community, they appear to operate consistently across the Adriatic farming grounds studied. The gill microbiome, in this sense, behaves like a host-associated community rather than a local environmental artifact. What did differ was time: the gill communities showed clear differences between months, indicating temporal dynamics in their composition over the course of the year-long sampling program.

The taxonomic analysis then delivered the study’s central insight. Within the family Endozoicomonadaceae, a group of Gammaproteobacteria well known from associations with marine invertebrates, the researchers found members without known relatives at the genus level, so-called unclassified Endozoicomonadaceae, present in the gills of mussels from both locations and throughout the entire study period. This persistence across space and time marks them as resident members of the gill microbiome, microbes that maintain a stable association with the host rather than drifting in and out with the seasons. Their consistent presence hints at a functional role, though the study, based on 16S rRNA gene profiles, does not directly demonstrate what that role might be.

In contrast, other members of the same family behaved very differently. Endozoicomonas and the taxon provisionally known as Candidatus Endoecteinascidia appeared in the gill microbiome of mussels from both locations only in certain months, marking them as transient taxa whose presence ebbs and flows with the seasons. The picture became even more site-specific with the Vibrionaceae. Vibrio and unclassified members of the Vibrionaceae were also transient, appearing only in certain months, but with a telling distinction: they were specific to Lim Bay. That a well-known group of bacteria, which includes both harmless marine organisms and significant pathogens, showed up as a seasonal, location-limited component of the gill community is likely to attract attention from aquaculture researchers, since Vibrio dynamics in farmed bivalves are a persistent concern for the industry.

The conceptual framework the study reinforces is one that microbiome science has been converging on for years: host-associated communities are mixtures of core and casual members, and only long-term, repeated sampling can tell them apart. A single snapshot of a gill microbiome might have recorded the unclassified Endozoicomonadaceae and concluded they were the whole story, or it might have caught a Vibrio bloom and drawn alarming conclusions. By sampling monthly across a year and pairing the mussel data with seawater and sediment references, the Croatian team was able to show that the gill community combines a stable backbone with a shifting overlay of seasonal and local arrivals. The temporal dynamics they observed, with community composition differing between months even as the overall gill signature remained distinct from the environment, suggest that both the host and the changing conditions of the Adriatic play a role in shaping who lives on the gills.

For the aquaculture sector, the implications are practical as well as conceptual. Mytilus galloprovincialis is farmed intensively in the Mediterranean, and the health of farmed mussels is increasingly understood in terms of their microbial associations. Knowing that a stable, resident core of Endozoicomonadaceae persists on the gills across sites and seasons provides a baseline against which future perturbations, whether from warming waters, disease outbreaks, or changes in farming practice, can be measured. Knowing that Vibrio appears only transiently and only at one of the two studied bays provides a template for monitoring programs that distinguish routine seasonal variation from genuine warning signs. And methodologically, the study demonstrates the value of the three-compartment design, comparing host, water, and sediment simultaneously, as a way to separate true host association from environmental background. As microbiome research moves from description toward prediction, studies of this kind, grounded in a full year of observation at real farming sites, provide the empirical foundation on which that transition will depend. The gills of the Mediterranean mussel, it turns out, are not just a filter but a curated habitat, one whose permanent residents and temporary guests can now be told apart.

Subject of Research: Resident and transient bacterial taxa in the gill microbiome of the Mediterranean mussel Mytilus galloprovincialis in the Adriatic Sea

Article Title: Resident and Transient Microbial Taxa in the Gill Microbiome of the Mussel Mytilus galloprovincialis

Article References: Korlević, M., Markovski, M., Kapetanović, D., Vardić Smrzlić, I., Orlić, K., Bolotin, J., Kožul, V., Bobanović-Ćolić, S., Nerlović, V., & Perić, L. (2026). Resident and Transient Microbial Taxa in the Gill Microbiome of the Mussel Mytilus galloprovincialis. Microbial Ecology. https://doi.org/10.1007/s00248-026-02888-y

Image Credits: AI Generated

DOI: 10.1007/s00248-026-02888-y

Keywords: gill microbiome, Mytilus galloprovincialis, Mediterranean mussel, Endozoicomonadaceae, Vibrionaceae, 16S rRNA gene sequencing, Adriatic Sea, aquaculture, marine microbiology, temporal dynamics, Gammaproteobacteria, microbial ecology