Benthic Crustacean Digestion Can Modulate the Environmental Fate of Microplastics in the Deep Sea.

Microplastics (MPs) are ubiquitous contaminants of the marine environment, and the deep seafloor is their ultimate sink compartment. Manipulative and field experiments provided evidence of the ingestion of MPs by deep-sea fauna, but knowledge of MPs' fate once ingested still remains scant. We provide evidence of MP partial retention and fragmentation mediated by digestion activity of a Norwegian langoustine, a good bioindicator for MP contamination of the deep sea. We report here that MPs in the intestines were more abundant and significantly smaller (up to 1 order of magnitude in surface) than those in the stomachs. Our results show that the stomach can act as a size-bottleneck for ingested MPs, enhancing the retention of larger particles within the stomach and promoting fragmentation into smaller plastic debris, which is then released in the intestine. Our results provide evidence that the langoustine is responsible for the fragmentation of MPs already accumulated in sediments through its scavenging activity and digestion. These findings highlight the existence of a new peculiar kind of "secondary" MPs, introduced in the environment by biological activities, which could represent a significant pathway of plastic degradation in a secluded and stable environment such as the deep sea.

Chronic and intensive bottom trawling impairs deep-sea biodiversity and ecosystem functioning.

Bottom trawling has many impacts on marine ecosystems, including seafood stock impoverishment, benthos mortality, and sediment resuspension. Historical records of this fishing practice date back to the mid-1300s. Trawling became a widespread practice in the late 19th century, and it is now progressively expanding to greater depths, with the concerns about its sustainability that emerged during the first half of the 20th century now increasing. We show here that compared with untrawled areas, chronically trawled sediments along the continental slope of the north-western Mediterranean Sea are characterized by significant decreases in organic matter content (up to 52%), slower organic carbon turnover (ca. 37%), and reduced meiofauna abundance (80%), biodiversity (50%), and nematode species richness (25%). We estimate that the organic carbon removed daily by trawling in the region under scrutiny represents as much as 60-100% of the input flux. We anticipate that such an impact is causing the degradation of deep-sea sedimentary habitats and an infaunal depauperation. With deep-sea trawling currently conducted along most continental margins, we conclude that trawling represents a major threat to the deep seafloor ecosystem at the global scale.

The challenge of proving the existence of metazoan life in permanently anoxic deep-sea sediments.

The demonstration of the existence of metazoan life in absence of free oxygen is one of the most fascinating and difficult challenges in biology. Danovaro et al. (2010) discovered three new species of the Phylum Loricifera, living in the anoxic sediments of the L'Atalante, a deep-hypersaline anoxic basin of the Mediterranean Sea. Multiple and independent analyses based on staining, incorporation of radiolabeled substrates, CellTracker Green incorporation experiments and ultra-structure analyses, allowed Danovaro et al. (2010) to conclude that these animals were able to spend their entire life cycle under anoxic conditions. Bernhard et al. (2015) investigated the same basin. Due to technical difficulties in sampling operations, they could not collect samples from the permanently anoxic sediment, and sampled only the redoxcline portion of the L'Atalante basin. They found ten individuals of Loricifera and provided alternative interpretations of the results of Danovaro et al. (2010). Here we analyze these interpretations, and present additional evidence indicating that the Loricifera encountered in the anoxic basin L'Atalante were actually alive at the time of sampling. We also discuss the reliability of different methodologies and approaches in providing evidence of metazoans living in anoxic conditions, paving the way for future investigations.This paper is a response to Bernhard JM, Morrison CR, Pape E, Beaudoin DJ, Todaro MA, Pachiadaki MG, Kormas KAr, Edgcomb VG. 2015. Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins. BMC Biology 2015 13:105.See research article at http://bmcbiol.biomedcentral.com/articles/10.1186/s12915-015-0213-6.

Effects of acidification on the biogeochemistry of unvegetated and seagrass marine sediments.

Many studies addressed ocean acidification (OA) effects on marine life, whereas its effects on sedimentary organic matter (OM) have received less attention. We investigated differences in OM features in sediments from unvegetated and seagrass (Posidonia oceanica) beds in a shallow hydrothermal area (Aeolian Archipelago, Mediterranean Sea), under natural (8.1-8.0) and acidified (7.8-7.9) conditions. We show that a pH difference of -0.3 units have minor effects on OM features in unvegetated sediments, but relevant consequences within acidified seagrass meadows, where OM quantity and nutritional quality are lower than those under natural pH conditions. Effects of acidified conditions on OM biogeochemistry vary between unvegetated and seagrass sediments, with lower C degradation rates and longer C turnover time in the former than in the latter. We conclude that OA, although with effects not consistent between unvegetated and vegetated sediments, can affect OM quantity, composition, and degradation, thus having possible far-reaching consequences for benthic trophic webs.

Effects of bottom trawling and environmental factors on benthic bacteria, meiofauna and macrofauna communities and benthic ecosystem processes.

Soft sediment marine benthic ecosystems comprise a diverse community of bacteria, meiofauna and macrofauna, which together support a range of ecosystem processes such as biogeochemical cycling. These ecosystems are also fishing grounds for demersal species that are often caught using bottom trawling. This fishing method can have deleterious effects on benthic communities by causing injury or mortality, and through alteration of sediment properties that in turn influence community structure. Although the impacts of bottom trawling on macrofauna are relatively well studied, less is known about the responses of meiofauna and bacteria to such disturbances, or how bottom trawling impacts benthic ecosystem processes. Quantifying trawling impacts against a background of natural environmental variability is also a challenge. To address these questions, we examined effects of bottom trawling and a range of environmental variables (e.g. water chemistry and physical and biochemical surface sediment properties) on a) bacterial, meiofaunal and macrofaunal community structure and b) benthic ecosystem processes (nutrient fluxes, extracellular enzyme activities and carbon turnover and degradation rates). We also investigated the link between the benthic macrofauna community and the same ecosystem processes. While there was a significant effect of bottom trawling intensity on macrofaunal community structure, the same was not seen for bacterial or meiofaunal community composition, which were more affected by environmental factors, such as surface sediment properties. The labile component of the surface sediment carbon pool was higher at highly trawled sites. Carbon degradation rates, extracellular enzyme activities, oxygen fluxes and some nutrient fluxes were significantly affected by trawling, but ecosystem processes were also strongly linked to the abundance of key bioturbators (Macoma balthica, Halicryptus spinulosus, Scoloplos armiger and Pontoporeia femorata). Although benthic ecosystems were affected by a combination of trawling and natural variability, disentangling these showed that the anthropogenic effects were clearest on the larger component of the community, i.e. macrofauna composition, and on ecosystem processes related to sedimentary carbon.

Sea cucumbers bioturbation potential outcomes on marine benthic trophic status under different temperature regimes.

Eutrophication affects coastal oceans worldwide, modifies primary production and sediment biogeochemistry and, overall, is progressively compromising marine ecosystems' integrity. Because of their known bioturbation ability, sea cucumbers are supposed to be candidates for mitigating benthic eutrophication. To provide insights on this, we investigated differences in organic matter quantity and biochemical composition (as proxies of benthic trophic status) of sediments and feces of the sea cucumber Holothuria tubulosa acclimated in mesocosms at temperatures comprised between natural conditions (14-26 °C) and an extreme of 29 °C (representing the highest anomaly under heat waves in the Mediterrranean Sea). Organic matter features differed significantly between sediments characterized by different trophic statuses and the holothuroid's feces, though with some exceptions. Feces resulted almost always organically enriched when compared with the ambient sediments, though with variable differences in composition in sediments characterized by different initial trophic status. Our results point out that sea cucumbers maintain their bioreactor capacity at all experimental temperatures including the (anomalous) highest one, irrespectively of the available food, suggesting that they could be profitably utilized to mitigate benthic eutrophication also in a warmer Mediterranean Sea.

Environmental Status and Geomorphological Characterisation of Seven Black Coral Forests on the Sardinian Continental Shelf (NW Mediterranean Sea).

Marine animal forests are key mesophotic ecosystems that are under threat from increasing natural and human pressures. Despite the fact that various international agreements strive to preserve these fragile ecosystems, the environmental status of the majority of these animal-structured environments is unknown. Assessing their environmental status is the first step needed to monitor these essential habitats' health over time and include them within conservation and protection frameworks, such as the Marine Strategy Framework Directive. Based on Multibeam data and ROV footage, we characterized the geomorphological setting and evaluated the environmental status of seven black coral forests in the centre of the Western Mediterranean Sea, using the Mesophotic Assemblages Conservation Status (MACS) Index. The presence of two antipatharians, Antipathella subpinnata and Leiopathes glaberrima, characterized the seven investigated sites, dwelling on rocky substrate characterized by different environmental drivers (i.e., depth, slope of the substrate, terrain ruggedness, topographic positioning index, and aspect). From the combined evaluation of the associated benthic community status and the anthropogenic impacts affecting it, a "high" and "good" environmental status was assessed for five out of the seven studied black forests, with only two forests classified as having a "moderate" and "poor" status, respectively. Overall, our study showed a site-specific variability of mesophotic black coral forest status, explained by different biological community structures and environmental conditions mainly associated with morphological and anthropogenic factors.

Rivers of waste: Anthropogenic litter in intermittent Sardinian rivers, Italy (Central Mediterranean).

While the increasing accumulation of anthropogenic litter in the marine environment has received considerable attention over the last decade, litter occurrence and distribution in rivers, the main source of marine litter, have been comparatively less investigated. Moreover, little information is available about the amount and typology of Riverine Anthropogenic Macro-litter (RAM) entering marine environments from intermittent rivers in low populated areas of the Mediterranean basin. To provide insights on this issue, we investigated density and composition of RAM accumulated over a total of 133 riverbanks, belonging to 37 river basins in the Sardinia Island (Mediterranean Sea). We report here that plastics, especially single-use items, represent the most frequent and abundant RAM category in all investigated basins. Statistical modelling revealed that occurrence of lightweight RAM (especially plastic) is mostly explained by levels of urban (12.3% of the relative contribution) and agricultural (12%) land use of the territory, whereas the proximity of bridges to the sampling point (21%) and the local population density (19.8%) are best predictors of heavy weighted RAM items (i.e., large metal items, appliances) occurrence. Our results confirm that plastics represent an important component of RAM and pinpoint that, beside plastic reduction policies and better waste management, actions aimed at abating and monitoring litter contamination should be localized on the proximity of bridges, whatever the local population density. Finally, to fill existing knowledge gaps in understanding the severity of litter discharge and accumulation in the Mediterranean Sea, land-to-sea systematic monitoring campaigns at appropriate spatial and temporal scales should be put in place.

Effects of Field Simulated Marine Heatwaves on Sedimentary Organic Matter Quantity, Biochemical Composition, and Degradation Rates.

Since rising temperature (T) will enhance biochemical reactions and coastal marine sediments are hotspots of carbon cycling, marine heatwaves' (MHWs') intensification caused by climate change will affect coastal biogeochemistry. We investigated the effects of MHWs on sediment organic matter (OM) in a nearshore locality (NW Sardinia, Mediterranean Sea) receiving an artificial warm water plume generating T anomalies of 1.5-5.0 °C. Sediments were collected before and after 3 and 11 weeks from the initial plume release. Both MHWs influenced sedimentary OM quantity, composition, and degradation rates, with major effects associated with the highest T anomaly after 3 weeks. Both MHWs enhanced sedimentary OM contents, with larger effects associated with the highest T anomaly. Phytopigment contents increased in the short term but dropped to initial levels after 11 weeks, suggesting the occurrence of thermal adaptation or stress of microphytobenthos. In the longer term we observed a decrease in the nutritional quality of OM and a slowdown of its turnover mediated by extracellular enzymes, suggestive of a decreased ecosystem functioning. We anticipate that intensification of MHWs will affect benthic communities not only through direct effects on species tolerance but also by altering benthic biogeochemistry and the efficiency of energy transfer towards higher trophic levels.

Detection of community-wide impacts of bottom trawl fishing on deep-sea assemblages using environmental DNA metabarcoding.

Although considerable research progress on the effects of anthropogenic disturbance in the deep sea has been made in recent years, our understanding of these impacts at community level remains limited. Here, we studied deep-sea assemblages of Sicily (Mediterranean Sea) subject to different intensities of benthic trawling using environmental DNA (eDNA) metabarcoding and taxonomic identification of meiofauna communities. Firstly, eDNA metabarcoding data did not detect trawling impacts using alpha diversity whereas meiofauna data detected a significant effect of trawling. Secondly, both eDNA and meiofauna data detected significantly different communities across distinct levels of trawling intensity when we examined beta diversity. Taxonomic assignment of the eDNA data revealed that Bryozoa was present only at untrawled sites, highlighting their vulnerability to trawling. Our results provide evidence for community-wide impacts of trawling, with different trawling intensities leading to distinct deep-sea communities. Finally, we highlight the need for further studies to unravel understudied deep-sea biodiversity.

Impact of resuspended mine tailings on benthic biodiversity and ecosystem processes: The case study of Portmán Bay, Western Mediterranean Sea, Spain.

Industrial seabed mining is expected to cause significant impacts on marine ecosystems, including physical disturbance and the generation of plumes of toxin-laden water. Portmán Bay (NW Mediterranean Sea), where an estimated amount of 60 Mt of mine tailings from sulphide ores were dumped from 1957 to 1990, is one of the most metal-polluted marine areas in Europe and worldwide. This bay can be used to assess the impact on marine ecosystems of particle settling from sediment plumes resulting from mine tailings resuspension. With this purpose in mind, we conducted a field experiment there to investigate subsequent effects of deposition of (artificially resuspended) contaminated sediments on (i) prokaryotic abundance and meiofaunal assemblages (in terms of abundance and diversity), (ii) the availability of trophic resources (in terms of organic matter biochemical composition), and (iii) a set of ecosystem functions including meiofaunal biomass, heterotrophic C production and C degradation rates. The results of this study show that mine tailings resuspension and plume deposition led to the decline of prokaryotic abundance and nematode's biodiversity. The later decreased because of species removal and transfer along with particle resuspension and plume deposition. Such changes were also associated to a decrease of the proteins content in the sediment organic matter, faster C degradation rates and higher prokaryotic C production. Overall, this study highlights that mine tailing resuspension and ensuing particle deposition can have deleterious effects on both prokaryotes and nematode diversity, alter biogeochemical cycles and accelerate C degradation rates. These results should be considered for the assessment of the potential effects of seabed mineral exploitation on marine ecosystems at large.

The first metazoa living in permanently anoxic conditions.

BACKGROUND: Several unicellular organisms (prokaryotes and protozoa) can live under permanently anoxic conditions. Although a few metazoans can survive temporarily in the absence of oxygen, it is believed that multi-cellular organisms cannot spend their entire life cycle without free oxygen. Deep seas include some of the most extreme ecosystems on Earth, such as the deep hypersaline anoxic basins of the Mediterranean Sea. These are permanently anoxic systems inhabited by a huge and partly unexplored microbial biodiversity. RESULTS: During the last ten years three oceanographic expeditions were conducted to search for the presence of living fauna in the sediments of the deep anoxic hypersaline L'Atalante basin (Mediterranean Sea). We report here that the sediments of the L'Atalante basin are inhabited by three species of the animal phylum Loricifera (Spinoloricus nov. sp., Rugiloricus nov. sp. and Pliciloricus nov. sp.) new to science. Using radioactive tracers, biochemical analyses, quantitative X-ray microanalysis and infrared spectroscopy, scanning and transmission electron microscopy observations on ultra-sections, we provide evidence that these organisms are metabolically active and show specific adaptations to the extreme conditions of the deep basin, such as the lack of mitochondria, and a large number of hydrogenosome-like organelles, associated with endosymbiotic prokaryotes. CONCLUSIONS: This is the first evidence of a metazoan life cycle that is spent entirely in permanently anoxic sediments. Our findings allow us also to conclude that these metazoans live under anoxic conditions through an obligate anaerobic metabolism that is similar to that demonstrated so far only for unicellular eukaryotes. The discovery of these life forms opens new perspectives for the study of metazoan life in habitats lacking molecular oxygen.

Foraging of the sea urchin Paracentrotus lividus (Lamarck, 1816) on invasive allochthonous and autochthonous algae.

Attempts to control marine invasive alien species (IAS) with native predators gained contrasting results, so far. To explore the feasibility of this approach to control the invasive marine alga Caulerpa cylindracea, we investigated the foraging behaviour of the sea urchin Paracentrotus lividus on three native macroalgae (Ulva sp., Penicillus capitatus and Cystoseira compressa) and on C. cylindracea. The consumption rate of C. cylindracea fresh biomass resulted larger than that of the other algae, when offered separately or in combination. C. cylindracea, however, was not the most attractive food item. The larger consumption rates of C. cylindracea can be explained by its specific caloric content (as assessed by its biochemical composition) that is lower than that of the other algae. Our results confirm that P. lividus can feed on C. cylindracea, but do not fully support its use to control C. cylindracea, unless in conditions where this alga is largely dominant because of other factors.

Colonization of plastic debris by the long-lived precious red coral Corallium rubrum: New insights on the "plastic benefits" paradox.

Seafloor macrolitter is ubiquitous in world's oceans; still, huge knowledge gaps exist on its interactions with benthic biota. We report here the colonization of plastic substrates by the Mediterranean red coral Corallium rubrum (L. 1758), occurring both in controlled conditions and in the wild at ca. 85 m depth in the Western Mediterranean Sea. Juveniles settled on seafloor macro-litter, with either arborescent or encrusting morphology, ranging from 0.6 to 3.5 mm in basal diameter and 0.2-7.1 years of age, also including a fraction (20%) of potentially sexually mature individuals. In controlled conditions, larvae settled and survived on plastic substrates for >60 days. Our insights show that marine plastic debris can provide favourable substrate for C. rubrum settlement either in controlled conditions or in the wild, suggesting their possible use in restoration activities. However, we pinpoint here that this potential benefit could result in adverse effects on population dynamics.

Microplastic pollution in perch (Perca fluviatilis, Linnaeus 1758) from Italian south-alpine lakes.

Microplastic particles (MPs) contamination of aquatic environments has raised a growing concern in recent decades because of their numerous potential toxicological effects. Although fish are among the most studied aquatic organisms, reports on MPs ingestion in freshwater environments are still scarce. Thus, there is still much to study to understand the uptake mechanisms, their potential accumulation among the food webs and their ecotoxicological effects. Here, MPs presence in the digestive system of one of the most widespread and commercially exploited freshwater fish, the perch (Perca fluviatilis, Linnaeus 1758), was investigated in four different south-alpine lakes, to assess the extent of ingestion and evaluate its relation to the body health condition. A total of 80 perch specimen have been sampled from the Italian lakes Como, Garda, Maggiore and Orta. Microplastic particles occurred in 86% of the analysed specimens, with average values ranging from 1.24 ± 1.04 MPs fish-1 in L. Como to 5.59 ± 2.61 MPs fish-1 in L. Garda. The isolated particles were mainly fragments, except in L. Como where films were more abundant. The most common polymers were polyethylene, polyethylene terephthalate, polyamide, and polycarbonate, although a high degree of degradation was found in 43% of synthetic particles, not allowing their recognition up to a single polymer. Despite the high number of ingested MPs, fish health (evaluated by means of Fulton's body condition and hepatosomatic index) was not affected. Instead, fullness index showed an inverse linear relationship with the number of ingested particles, which suggests that also in perch MPs presence could interfere with feeding activity, as already described for other taxa.

Mediterranean rocky reefs in the Anthropocene: Present status and future concerns.

Global change is striking harder and faster in the Mediterranean Sea than elsewhere, where high levels of human pressure and proneness to climate change interact in modifying the structure and disrupting regulative mechanisms of marine ecosystems. Rocky reefs are particularly exposed to such environmental changes with ongoing trends of degradation being impressive. Due to the variety of habitat types and associated marine biodiversity, rocky reefs are critical for the functioning of marine ecosystems, and their decline could profoundly affect the provision of essential goods and services which human populations in coastal areas rely upon. Here, we provide an up-to-date overview of the status of rocky reefs, trends in human-driven changes undermining their integrity, and current and upcoming management and conservation strategies, attempting a projection on what could be the future of this essential component of Mediterranean marine ecosystems.

Potentially combined effect of the invasive seaweed Caulerpa cylindracea (Sonder) and sediment deposition rates on organic matter and meiofaunal assemblages.

The seaweed Caulerpa cylindracea (Sonder) is one of the most successful marine bioinvaders worldwide. Caulerpa cylindracea can influence the quantity and biochemical composition of sedimentary organic matter (OM). However, it is still unknown if the effects of C. cylindracea on both OM and small metazoans (i.e. meiofauna) can change according to different sediment deposition rates. To provide insights on this, we investigated the biochemical composition of sediments along with the abundance and composition of meiofaunal assemblages in sediments colonized and not-colonized by the seaweed C. cylindracea under different regimes of sediment deposition. Our results show that the presence of the invasive alga C. cylindracea could alter quantity, biochemical composition, and nutritional quality of organic detritus and influence the overall functioning of the benthic system, but also that the observed effects could be context-dependent. In particular, we show that the presence of C. cylindracea could have a positive effect on meiofaunal abundance wherever the sediment deposition rates are low, whereas the contextual presence of high to medium sedimentation rates can provoke an accumulation of sedimentary organic matter, less favourable bioavailability of food for the benthos, and consequent negative effects on meiofauna.

Impact of historical sulfide mine tailings discharge on meiofaunal assemblages (Portmán Bay, Mediterranean Sea).

Portmán Bay is one of the most contaminated and chronically impacted coastal marine areas of the world. Here, from the 1957 to 1990, about 60 million tons of mine tailings from the processing of sulfide ores were dumped directly at the shoreline. The resulting deposit provides a unique opportunity to assess the impact of mine tailings on coastal marine ecosystems after ca 30 years since the discharge has ceased. We investigated meiofaunal abundance, biomass and biodiversity along a gradient of metal concentration that overlaps with a bathymetric gradient from 30 to 60 m depth. Despite the localized presence of extremely high concentration of metals, the bay was not a biological desert, but, nevertheless, was characterized by evident signs of impact on benthic diversity. Meiofaunal variables increased significantly with decreasing metal contamination, eventually reaching values comparable to other uncontaminated coastal sediments. Our results show that mine tailings influenced the spatial distribution of meiofaunal taxa and nematode species composition. In particular, we report here that the bay was characterized by the dominance of nematode opportunistic species tolerant to high metal concentration. The effects of mine tailing discharge on meiofaunal biodiversity and composition were still evident ca 30 years after the end of the mining activities. Overall, this study provides new insights on the potential impact of mine tailings disposal and metal contamination in coastal sediments, and, can also contribute to predict the potential long-term consequences of ever-expanding deep-sea mining industry on benthic environments.

Denitrification, Nitrogen Uptake, and Organic Matter Quality Undergo Different Seasonality in Sandy and Muddy Sediments of a Turbid Estuary.

The interaction between microbial communities and benthic algae as nitrogen (N) regulators in poorly illuminated sediments is scarcely investigated in the literature. The role of sediments as sources or sinks of N was analyzed in spring and summer in sandy and muddy sediments in a turbid freshwater estuary, the Curonian Lagoon, Lithuania. Seasonality in this ecosystem is strongly marked by phytoplankton community succession with diatoms dominating in spring and cyanobacteria dominating in summer. Fluxes of dissolved gas and inorganic N and rates of denitrification of water column nitrate (Dw) and of nitrate produced by nitrification (Dn) and sedimentary features, including the macromolecular quality of organic matter (OM), were measured. Shallow/sandy sites had benthic diatoms, while at deep/muddy sites, settled pelagic microalgae were found. The OM in surface sediments was always higher at muddy than at sandy sites, and biochemical analyses revealed that at muddy sites the OM nutritional value changed seasonally. In spring, sandy sediments were net autotrophic and retained N, while muddy sediments were net heterotrophic and displayed higher rates of denitrification, mostly sustained by Dw. In summer, benthic oxygen demand increased dramatically, whereas denitrification, mostly sustained by Dn, decreased in muddy and remained unchanged in sandy sediments. The ratio between denitrification and oxygen demand was significantly lower in sandy compared with muddy sediments and in summer compared with spring. Muddy sediments displayed seasonally distinct biochemical composition with a larger fraction of lipids coinciding with cyanobacteria blooms and a seasonal switch from inorganic N sink to source. Sandy sediments had similar composition in both seasons and retained inorganic N also in summer. Nitrogen uptake by microphytobenthos at sandy sites always exceeded the amount loss via denitrification, and benthic diatoms appeared to inhibit denitrification, even in the dark and under conditions of elevated N availability. In spring, denitrification attenuated N delivery from the estuary to the coastal area by nearly 35%. In summer, denitrification was comparable (~100%) with the much lower N export from the watershed, but N loss was probably offset by large rates of N-fixation.

Archaeal diversity in deep-sea sediments estimated by means of different terminal-restriction fragment length polymorphisms (T-RFLP) protocols.

Despite the increasing recognition of the quantitative importance of Archaea in all marine systems, the protocols for a rapid estimate of Archaeal diversity patterns in deep-sea sediments have been only poorly tested yet. Sediment samples from 11 deep-sea sites (from 79 degrees N to 36 degrees N, at depths comprised from 469 to 5,571 m) were used to compare the performance of two different primer sets (ARCH21f/ARCH958r and ARCH109f/ARCH 915r) and three restriction enzymes (AluI, Rsa I, and HaeIII) for the fingerprinting analysis of Archaeal diversity using terminal-restriction fragment length polymorphisms (T-RFLP). In silico and experimental analyses indicated that different combinations of primer sets and restriction enzymes provided different values of benthic Archaeal ribotype richness and different Archaeal assemblage compositions. The use of the ARCH109f/ARCH 915r primer set in combination with AluI provided the best results (a number of ribotypes up to four folds higher than other combinations), suggesting that this primer set should be used in future studies dealing with the analysis of the patterns of Archaeal diversity in deep-sea sediments. Multivariate multiple regression analysis revealed that, whatever the T-RFLP protocol utilized, latitude and temperature explained most of the variance in benthic Archaeal ribotype richness, while water depth had a negligible role.