David Grémillet
email: david.gremillet@cefe.cnrs.fr

Sébastien Descamps
email: sebastien.descamps@npolar.no

Ecological impacts of climate change on Arctic marine megafauna Authors

Keywords: Biogeography, Citizen science, Global change, Long-term monitoring, Oceanography, Polar

Global warming affects the Arctic more than any other region. Mass media constantly relay apocalyptic visions of climate change threatening Arctic wildlife, especially emblematic megafauna such as polar bears, whales and seabirds. Yet, we are just beginning to understand such ecological impacts on marine megafauna at the scale of the Arctic. This knowledge is geographically and taxonomically biased, with striking deficiencies in the Russian Arctic and strong focus on exploited species such as cod. Beyond a synthesis of scientific advances in the last five years, we provide ten key questions to be addressed by future work, and outline the requested methodology. This framework builds upon long-term Arctic monitoring inclusive of local communities, whilst capitalizing on high-tech and big data approaches.

Climate change and Arctic marine megafauna

The Arctic is warming nearly four times faster than the rest of the planet, overshooting predictions [START_REF] Rantanen | The Arctic has warmed nearly four times faster than the globe since 1979[END_REF].

These exceptional trends are due to Arctic amplification (see glossary) [START_REF] Previdi | Arctic amplification of climate change: a review of underlying mechanisms[END_REF], and abiotic consequences are manifold. Those include enhanced precipitation, sea surface temperatures and storminess, a declining cryosphere (land and sea ice, permafrost), intensified hydrological cycles, and coastal erosion [START_REF] Box | Key indicators of Arctic climate change: 1971-2017[END_REF]. Such changes affect ocean circulation, both at local and global scales, with feedback effects on atmospheric circulation, extreme weather events and sea level rise, also at lower latitudes [START_REF] Henderson | Local and Remote Atmospheric Circulation Drivers of Arctic Change: A Review[END_REF][START_REF] Moon | The expanding footprint of rapid Arctic change[END_REF]. In addition, a vanishing cryosphere leads to the release of chemicals and plastics, some of them toxic [START_REF] Sonne | Emerging contaminants and biological effects in Arctic wildlife[END_REF],

and to increased anthropogenic activities also contributing to enhanced pollution [START_REF] Svavarsson | Pollutants from shipping-new environmental challenges in the subarctic and the Arctic Ocean[END_REF].

The Arctic is ca. 67% a marine region, and climatic changes have profound abiotic effects on aquatic ecosystems [START_REF] Post | The polar regions in a 2 C warmer world[END_REF], notably through a transformed light environment following the disappearance of seaice, changes in ocean stratification, acidification, enhanced nutrient fluxes from land to sea and bentho-pelagic coupling, as well as shifting haloclines (salinity stratification) [START_REF] Box | Key indicators of Arctic climate change: 1971-2017[END_REF]9,[START_REF] Lewis | Changes in phytoplankton concentration now drive increased Arctic Ocean primary production[END_REF]. From a biotic point of view, Arctic marine food webs are rapidly transformed by the spread of northern temperate species, leading to a borealisation of the Arctic [START_REF] Ingvaldsen | Physical manifestations and ecological implications of Arctic Atlantification[END_REF]. Warming also opens the door to new pathogens, parasites and non-indigenous species [START_REF] Delgado-Baquerizo | The proportion of soil-borne pathogens increases with warming at the global scale[END_REF][START_REF] Chan | Climate change opens new frontiers for marine species in the Arctic: Current trends and future invasion risks[END_REF], and enhances connectivity, for instance through new species dispersal between the Pacific and Atlantic oceans, across the Arctic basin [START_REF] Clairbaux | Climate change could overturn bird migration: Transarctic flights and high-latitude residency in a sea ice free Arctic[END_REF]. The architecture of food webs is thereby transformed, through new predator-prey relationships, new competitors and shifting phenologies [START_REF] Florko | Predicting how climate change threatens the prey base of Arctic marine predators[END_REF]. In this context, one of the most prominent changes is the modified spatiotemporal occurrence of algal blooms, potentially leading to a 'marine greening of the Arctic' [START_REF] Lewis | Changes in phytoplankton concentration now drive increased Arctic Ocean primary production[END_REF], but also to new harmful algal blooms [START_REF] Anderson | Evidence for massive and recurrent toxic blooms of Alexandrium catenella in the Alaskan Arctic[END_REF]. These shifts reverberate across Arctic marine biophysical systems and those are currently entering new, unprecedented states [START_REF] Box | Key indicators of Arctic climate change: 1971-2017[END_REF].

Within such rapidly changing landscapes, Arctic marine megafauna (Figure 1) and First Nations relying on traditional food sources, seem to share a destiny [START_REF] Grose | Climate change will re-draw the map for marine megafauna and the people who depend on them[END_REF]. Marine megafauna are defined as all cnidarian (e.g. jellyfish), mollusks (e.g. squid), fishes, reptiles, birds and mammals which are larger than other marine species and/or are predators playing a key functional role in food webs [START_REF] Moleón | Rethinking megafauna[END_REF]. With the exception of peoples of the reindeer, Arctic First Nations are coastal, and tightly linked to the aquatic environment and its resources [START_REF] Hauser | Co-production of knowledge reveals loss of Indigenous hunting opportunities in the face of accelerating Arctic climate change[END_REF]. Marine megafauna are therefore economically and culturally essential for Arctic peoples, as food base and key elements of founding narratives. Both these elements foster the resilience of Arctic First Nations, in the past and when facing current global change [START_REF] Nuttall | Indigenous peoples, self-determination and the Arctic environment[END_REF]. For instance, the narwhal (Monodon monoceros) is hunted from Alaska to Greenland, and its skin highly valued as traditional food. The species is also subjected to a legend, told across the Arctic in slightly different versions, according to which the narwhal's tusk is made of the rolled-up hair of a drowned woman. Close relatedness between Arctic people and marine megafauna is also underlined by the panarctic legend of the mother of the sea, a woman who rules over all marine animals and is married to a seabird, the northern fulmar (Fulmarus glacialis).

As keystone species at the apex of food chains, Arctic marine megafauna integrate underlying processes: Their food base depends upon marine productivity, and they are also exposed to contaminants bio-accumulated across trophic levels [START_REF] Townhill | Pollution in the Arctic Ocean: An overview of multiple pressures and implications for ecosystem services[END_REF]. In this context, marine megafauna not only function as ecological indicators providing information about the state of marine ecosystems, they may also become flagship organisms motivating decision makers to act for nature conservation [START_REF] Lescroël | Seeing the ocean through the eyes of seabirds: A new path for marine conservation?[END_REF].

For these different reasons, it is essential to better understand the ecological impacts of climate change on Arctic marine megafauna, and knowledge has been recently gathered in this matter. We review these insights and assess whether they are sufficient to test the impacts of Arctic climate change on ecological processes affecting marine megafauna. To guide future work, we outline ten key research questions, and provide a novel, integrative research framework and methodological toolkit. This rationale blends a wide range of techniques, including long-term monitoring, emerging technologies and citizen science programs.

A synthesis of ecological impacts

We reviewed recent existing knowledge on the ecological impacts of climate change on Arctic marine megafauna. We thereby focused on the five years following the Paris Agreement under the United Nations Framework Convention on Climate Change, which is considered a major landmark for international awareness on climate change impacts. To this end, we searched the Web of Knowledge (in English) and CyberLeninka (in Russian) in February-April 2022, focusing on climate change-related articles published since 2017 on fish, jellyfish, squid, seabirds and marine mammals (see details in Supplementary information 1). A focus on scientific knowledge of the past five years also follows the guidelines for reviews in Trends in Ecology and Evolution. We acknowledge the fact that this may omit some previous work, partly compensated by 25 years of polar research and knowledge by the two authors. Moreover, our conclusions are supported by former reviews focused on Arctic marine ecosystem functioning [START_REF] Wassmann | Footprints of climate change in the Arctic marine ecosystem[END_REF]. Finally, since Arctic climate change mainly accelerated in recent years [START_REF] Rantanen | The Arctic has warmed nearly four times faster than the globe since 1979[END_REF], it seemed appropriate to focus on investigations conducted during this specific period.

Our analysis yielded 250 relevant publications (Suppl. 1). Fifty percent of the studies using empirical data (n=173) were based on > 11 years of data and 17% on ≥30 years of data (maximum 131 years, [START_REF] Hansen | Centennial relationships between ocean temperature and Atlantic puffin production reveal shifting decennial trends[END_REF]). Overall, there was a major focus on fish (36% of all studies). The analysis also revealed a strong bias towards a limited number of species: 40% of fish studies focused on Atlantic and polar cod (Gadus morhua and Boreogadus saida, respectively), and 20% (n=49) of all studies dealt with climate change impacts on polar bears (Ursus maritimus). These species were the most studied because they are either of high commercial value (Atlantic cod), play a key role in ecosystem functioning (polar cod), or because they are emblematic of the Arctic (polar bear). A relatively large number of fish species were considered (>80), because many studies were based on survey tows (e.g. 82 species included in [START_REF] Campana | Shifting fish distributions in warming sub-Arctic oceans[END_REF]).

Very few studies were conducted on cephalopods (4 in total, including 2 reviews), and the two most abundant species in the Arctic, i.e. Rossa palpebrosa and Gonatus fabricii were the focus of only two case studies [START_REF] Golikov | Diet and life history reduce interspecific and intraspecific competition among three sympatric Arctic cephalopods[END_REF][START_REF] Golikov | First assessment of biomass and abundance of cephalopods Rossia palpebrosa and Gonatus fabricii in the Barents Sea[END_REF]. Nineteen species of marine mammals were studied, with the beluga (or white) whale Delphinapterus leucas being the second most-studied species (n=13 studies) after the polar bear. Forty-four seabird species were studied, but many were part of general at-sea surveys and not the direct focus of the work. The thick-billed murre (Uria lomvia) was the most-studied seabird and appeared in 52% (n=27) of all seabird-related publications.

Most of the studies dealt with six main topics (Table 1). The relative importance of each topic varied among guilds, but spatial distribution/habitat use and individual state (body condition, growth, physiological state) were the most common topics for fish, seabirds and marine mammals. Despite the potential importance of diet shifts for marine megafauna population dynamics, very few studies investigated fish or seabird diets. Finally, whatever the guild considered, very few studies addressed changes at the community or ecosystem level (Table 1).

We also identified a strong geographical bias (Figure 2): the most intensively studied areas were US Alaskan waters as defined by the country's exclusive economic zone (136 studies per million km 2 ), followed by Norwegian waters (30 studies per million km 2 ) and, to a lesser extent, Canadian, Greenlandic and Icelandic waters [START_REF] Delgado-Baquerizo | The proportion of soil-borne pathogens increases with warming at the global scale[END_REF][START_REF] Ingvaldsen | Physical manifestations and ecological implications of Arctic Atlantification[END_REF] and 9 studies per million km 2 , respectively). The analysis stressed the deficit of studies within Russian waters (6 studies per million km 2 ), with the exception of the Barents Sea where research by Norwegian and Russians scientists is leading to advanced ecological understanding.

Therefore, the first prominent conclusion of our review is that existing knowledge on climate change impacts on Arctic marine megafauna is extremely biased with respect to studied species and geographical coverage. On the basis of this limited knowledge, our current understanding of ecological processes at the individual-, populational-, ecosystem-and landscape-level can be summarized as follows:

Physiological and behavioral responses

While this was a major research focus in the past [START_REF] Scholander | Body insulation of some arctic and tropical mammals and birds[END_REF], morphological and physiological adaptation to Arctic climates has been critically understudied in recent years [START_REF] Ste-Marie | A first look at the metabolic rate of Greenland sharks (Somniosus microcephalus) in the Canadian Arctic[END_REF]. Well-insulated Arctic endotherms such as murres (Uria sp.) may save energy in warmer winter conditions [START_REF] Clairbaux | Meeting Paris agreement objectives will temper seabird winter distribution shifts in the North Atlantic Ocean[END_REF], but easily overheat in summer [START_REF] Choy | Limited heat tolerance in a cold-adapted seabird: implications of a warming Arctic[END_REF], with consequences for their water and energy balance, and potential casualties during heat waves. In ectothermic fish, higher temperatures boost metabolic rates, but the gill-oxygen limitation theory predicts that warmer Arctic waters will contain less oxygen, therefore limiting growth and maximum size of water-breathing organisms [START_REF] Pauly | The gill-oxygen limitation theory (GOLT) and its critics[END_REF]. Warming also goes along with acidification of Arctic waters [START_REF] Terhaar | Emergent constraint on Arctic Ocean acidification in the twenty-first century[END_REF]: In Atlantic cod (Gadus morhua) and polar cod (Boreogadus saida), such acidification impairs adult swimming capacity [START_REF] Kunz | Aerobic capacities and swimming performance of polar cod (Boreogadus saida) under ocean acidification and warming conditions[END_REF], and causes a narrowing of embryonic thermal ranges, with a potentially critical impact on juvenile fitness [START_REF] Dahlke | Northern cod species face spawning habitat losses if global warming exceeds 1.5[END_REF]. Further synergetic adverse effects of Arctic warming involve enhanced zoonotic pathogen exposure, for instance in polar bears [START_REF] Pilfold | Long-term increases in pathogen seroprevalence in polar bears (Ursus maritimus) influenced by climate change[END_REF], as well as contamination by chemical pollutants [START_REF] Bender | Combined effects of crude oil exposure and warming on eggs and larvae of an arctic forage fish[END_REF] and plastics [START_REF] Bergmann | Plastic pollution in the Arctic[END_REF]. In this context, Arctic ecotoxicology is currently booming [START_REF] Albert | Seasonal variation of mercury contamination in Arctic seabirds: a panarctic assessment[END_REF], yet with a limited number of analyses testing fitness consequences of contaminant exposure [START_REF] Amélineau | Arctic climate change and pollution impact little auk foraging and fitness across a decade[END_REF].

In contrast to the paucity of physiological studies investigating the consequences of Arctic warming for marine megafauna, most recent publications focused on behavioral adjustments to rapidly changing environmental conditions, notably the disappearance of sea-ice and shifting prey distributions [START_REF] Jansen | Larval drift dynamics, thermal conditions and the shift in juvenile capelin distribution and recruitment success around Iceland and East Greenland[END_REF].

These investigations confirmed the pivotal role of flexible foraging behavior, enabling some marine species to buffer the consequences of Arctic climate change [START_REF] Grémillet | Little auks buffer the impact of current Arctic climate change[END_REF][START_REF] Hamilton | Contrasting changes in space use induced by climate change in two Arctic marine mammal species[END_REF]. For instance, long-term stable isotopic analyses showed dietary shifts in beluga whales (Delphinapterus leucas), ringed seals (Pusa hispida), Greenland halibut (Reinhardtius hippoglossoides) and anadromous Arctic char (Salvelinus alpinus) from Cumberland Sound, Nunavut [START_REF] Yurkowski | A temporal shift in trophic diversity among a predator assemblage in a warming Arctic[END_REF]. Such trophic re-arrangements may affect the architecture of entire food webs [START_REF] Breed | Sustained disruption of narwhal habitat use and behavior in the presence of Arctic killer whales[END_REF][START_REF] Griffith | Ecological resilience of Arctic marine food webs to climate change[END_REF]. Foraging plasticity also triggers shifting foraging habitats, such as for ringed seal (Pusa hispida) in Hudson Bay [START_REF] Florko | Predicting how climate change threatens the prey base of Arctic marine predators[END_REF], and ultimately lead to community-wide northward shifts, as recorded for seabirds in the Northern Bering and Chukchi seas [START_REF] Kuletz | Distributional shifts among seabird communities of the Northern Bering and Chukchi seas in response to ocean warming during 2017-2019[END_REF]. Finally, fish communities may also seek deeper habitats as the sea warms [START_REF] Burrows | Ocean community warming responses explained by thermal affinities and temperature gradients[END_REF]. Spatial re-arrangements may occur during residency, as well as during migration, potentially leading to the colonization of new habitats and enhancing the likelihood of speciation events [START_REF] Clairbaux | Climate change could overturn bird migration: Transarctic flights and high-latitude residency in a sea ice free Arctic[END_REF]. As sea-ice habitats vanish, recent work stressed the importance of coastal glacier fronts as refugia attracting Arctic fish [START_REF] Bouchard | West Greenland ichthyoplankton and how melting glaciers could allow Arctic cod larvae to survive extreme summer temperatures[END_REF], birds and mammals [START_REF] Stempniewicz | Marine birds and mammals foraging in the rapidly deglaciating Arctic fjord-numbers, distribution and habitat preferences[END_REF]. Those lead to lower temperatures and localized upwelling enhancing prey availability [START_REF] Bertrand | Fine-scale spatial segregation in a pelagic seabird driven by differential use of tidewater glacier fronts[END_REF], but also promote predator contamination by pollutants and plastics [START_REF] Collard | Plastic ingestion by Arctic fauna: A review[END_REF]. Yet, spatial and trophic plasticity is likely more the exception than the norm in Arctic marine megafauna, especially in long-lived species such as marine mammals, seabirds and some fishes. This is due to the strong repeatability and persistence of their foraging behavior, of their marked philopatry [START_REF] Harris | Personality predicts foraging site fidelity and trip repeatability in a marine predator[END_REF], and to the lack of alternative habitats for the northernmost species, which cannot shift further poleward. Overall, recent big data approaches based on biologging triggered major advances in our understanding of behavioral and ecophysiological responses of marine megafauna to Arctic warming [START_REF] Davidson | Ecological insights from three decades of animal movement tracking across a changing Arctic[END_REF], but only a minority of those infer fitness consequences [START_REF] Reimer | Modeling optimal responses and fitness consequences in a changing Arctic[END_REF]. In this context, assessing the energy balance of animals facing environmental change by linking information on their foraging behavior and their energetics, appears as a major avenue to understand individual responses and their fitness costs [START_REF] Pagano | High-energy, high-fat lifestyle challenges an Arctic apex predator, the polar bear[END_REF].

Demographic and populational impacts

The effect of climate change on Arctic megafauna demography and population trajectories remains largely unknown, especially for marine mammals (Table 1). A few studies on fish [START_REF] Bouchard | Climate warming enhances polar cod recruitment, at least transiently[END_REF][START_REF] Yasumiishi | Climate-related changes in the biomass and distribution of small pelagic fishes in the eastern Bering Sea during late summer, 2002-2018[END_REF], seabirds [START_REF] Gutowsky | Sea ice extent and phenology influence breeding of high-Arctic seabirds: 4 decades of monitoring in Nunavut, Canada[END_REF][START_REF] Descamps | Species and spatial variation in the effects of sea ice on Arctic seabird populations[END_REF] or mammals [START_REF] Chambault | The impact of rising sea temperatures on an Arctic top predator, the narwhal[END_REF] identified significant relationships between population abundance and environmental stressors associated to climate change (e.g. sea surface temperature, timing of sea-ice break-up) based on empirical long-term data. Several others identified long-term trends in megafauna populations [START_REF] Strøm | Population status and trend of the threatened ivory gull Pagophila eburnea in Svalbard[END_REF][START_REF] Florko | Decreasing sea ice conditions in western Hudson Bay and an increase in abundance of harbour seals (Phoca vitulina) in the Churchill River[END_REF] and explained these trends in relation to climate change, but without formally testing for such associations. The small number of such studies is a direct consequence of the paucity in long-term time-series on Arctic megafauna population size. However, even in the absence of empirical abundance data, approaches based on traditional ecological knowledge [START_REF] Henri | Inuit knowledge of Arctic Terns (Sterna paradisaea) and perspectives on declining abundance in southeastern Hudson Bay, Canada[END_REF] or modelling may help understanding how Arctic populations are, or will be, responding to climate change [START_REF] Clairbaux | Climate change could overturn bird migration: Transarctic flights and high-latitude residency in a sea ice free Arctic[END_REF][START_REF] Razgour | An integrated framework to identify wildlife populations under threat from climate change[END_REF][START_REF] Heath | Ecosystem approach to harvesting in the Arctic: Walking the tightrope between exploitation and conservation in the Barents Sea[END_REF][START_REF] Reimer | Ringed seal demography in a changing climate[END_REF][START_REF] Bårdsen | Multiple stressors: modeling the effect of pollution, climate, and predation on viability of a sub-arctic marine bird[END_REF].

A common alternative consists in looking at effects on single demographic parameters, to infer the potential development of a given population [START_REF] Hansen | Centennial relationships between ocean temperature and Atlantic puffin production reveal shifting decennial trends[END_REF][START_REF] Bender | Combined effects of crude oil exposure and warming on eggs and larvae of an arctic forage fish[END_REF][START_REF] Rode | Seal body condition and atmospheric circulation patterns influence polar bear body condition, recruitment, and feeding ecology in the Chukchi Sea[END_REF]. For example, early sea-ice break-up and longer ice-free periods led to smaller litter size for polar bears in Baffin Bay (between Canada and Greenland), suggesting a negative effect of Arctic warming on the Baffin Bay polar bear population [START_REF] Laidre | Interrelated ecological impacts of climate change on an apex predator[END_REF]. However, such results should be interpreted with caution. Indeed, links between changes in a single demographic parameter and changes in population growth rate also depend on the sensitivity of population growth rate with respect to this parameter [START_REF] De Kroon | Elasticity: the relative contribution of demographic parameters to population growth rate[END_REF] and changes in other demographic parameters. Thereby, different parameters may show antagonistic responses to environmental change [START_REF] Cleasby | Climatic conditions produce contrasting influences on demographic traits in a long-distance Arctic migrant[END_REF], and analyses integrating the response of multiple demographic parameters are needed to understand population dynamics. Ideally, these analyses should also consider environmental conditions throughout the life-cycle, as many megafauna species are migratory and do not stay in the Arctic all year round. Environments encountered on the winter grounds may be of paramount importance in driving population dynamics, as highlighted >50 years ago by D. Lack [START_REF] Lack | Population studies of birds[END_REF] ,and should also be incorporated in demographic studies.

Overall, whatever the approach used, no general conclusion can be drawn yet: climate warming may have positive, negative or no effects on the vital rates and/or population trajectories of Arctic megafauna depending on the species and/or region considered. Endemic Arctic species may respond more negatively to climate warming [START_REF] Burrows | Ocean community warming responses explained by thermal affinities and temperature gradients[END_REF][START_REF] Descamps | As the Arctic becomes boreal: ongoing shifts in a high-Arctic seabird community[END_REF] but this is not an absolute pattern either. For example, the biomass of the polar cod, a true Arctic species, in the Canadian Arctic was positively correlated to sea-surface temperature [START_REF] Bouchard | Climate warming enhances polar cod recruitment, at least transiently[END_REF]. Beyond interspecific variation in climate change response, it is also essential to consider spatial variation in intraspecific responses. For example, relationships between sea-ice extent and the colony size of black-legged kittiwakes varied among fjords in Svalbard, potentially due to contrasting local oceanographic conditions [START_REF] Descamps | Species and spatial variation in the effects of sea ice on Arctic seabird populations[END_REF]. Also, loss of sea-ice had negative effects on polar bears in Baffin Bay [START_REF] Laidre | Interrelated ecological impacts of climate change on an apex predator[END_REF], but these effects were null or positive in the Chukchi Sea [START_REF] Rode | Seal body condition and atmospheric circulation patterns influence polar bear body condition, recruitment, and feeding ecology in the Chukchi Sea[END_REF].

Such spatial variation in the effects of climate warming may reflect local variability in other

environmental parameters [START_REF] Suggitt | Extinction risk from climate change is reduced by microclimatic buffering[END_REF]. Alternatively, they may also be the consequence of potentially crucial, but often overlooked non-linear effects. For instance, polar bears in the Chukchi Sea maintained body condition despite vanishing sea-ice [START_REF] Rode | Seal body condition and atmospheric circulation patterns influence polar bear body condition, recruitment, and feeding ecology in the Chukchi Sea[END_REF], contrary to bears from the Bering Sea whose condition declined [START_REF] Laidre | Interrelated ecological impacts of climate change on an apex predator[END_REF]. These findings suggesting that Chukchi Sea bears are not currently limited by sea ice [START_REF] Rode | Seal body condition and atmospheric circulation patterns influence polar bear body condition, recruitment, and feeding ecology in the Chukchi Sea[END_REF] may be due to non-linearity between sea-ice conditions and polar bear life history. Notably, in the Chukchi Sea, sea-ice cover may still be above the threshold below which a declining sea ice has detrimental effects [START_REF] Rode | Seal body condition and atmospheric circulation patterns influence polar bear body condition, recruitment, and feeding ecology in the Chukchi Sea[END_REF]. The concept of threshold, or tipping point [START_REF] Heinze | The quiet crossing of ocean tipping points[END_REF][START_REF] Duarte | Tipping elements in the Arctic marine ecosystem[END_REF], thereby remains essential [START_REF] May | Thresholds and breakpoints in ecosystems with a multiplicity of stable states[END_REF]. Even if the increase in temperature is more or less a linear process, it is associated with non-linear changes in other climatic parameters (e.g. snowfall, [START_REF] Bintanja | The impact of Arctic warming on increased rainfall[END_REF]). Equally, the behavioural, physiological and population responses to all these numerous and complex environmental changes have no reason to be linear [START_REF] Hansen | Centennial relationships between ocean temperature and Atlantic puffin production reveal shifting decennial trends[END_REF][START_REF] Descamps | Circumpolar dynamics of a marine top-predator track ocean warming rates[END_REF].

Overall, species-specific and spatial variation in climate change effects, combined with potential nonlinearity in these effects, hinders our capacity to make general predictions regarding Arctic megafauna population trajectories. One key limiting factor is the lack of long-term abundance data for most species and/or regions.

Impacts on biodiversity and ecosystem functioning

Predicting how biodiversity responds to climate change [START_REF] Bellard | Impacts of climate change on the future of biodiversity[END_REF] and how changes in biodiversity affect ecosystem functioning [START_REF] Cardinale | Biodiversity loss and its impact on humanity[END_REF] are active fields of research. However, very few studies on Arctic megafauna have been undertaken in these specific areas. Our literature review identified only one that has explicitly assessed the impact of climate change on Arctic megafauna biodiversity. This study [START_REF] Mérillet | Fish Assemblages of a Sub-Arctic Fjord Show Early Signals of Climate Change Response Contrary to the Benthic Assemblages[END_REF] found positive global warming effect on fish biodiversity in an Arctic fjord in Northern Norway, whereby species richness and Shannon diversity of fishes increased following the arrival of warmwater species. Other studies focused on species composition [START_REF] Descamps | As the Arctic becomes boreal: ongoing shifts in a high-Arctic seabird community[END_REF][START_REF] Mueter | Changes in diversity and species composition across multiple assemblages in the Eastern Chukchi Sea during two contrasting years are consistent with borealization[END_REF][START_REF] Gall | Ecological shift from piscivorous to planktivorous seabirds in the Chukchi Sea, 1975-2012[END_REF], as a metric for species richness. These findings generally support the borealisation of the Arctic marine environment, with north temperate (or "boreal) species becoming more abundant. Species richness represents, however, only one facet of biodiversity [START_REF] Cardinale | Biodiversity loss and its impact on humanity[END_REF] so that reported changes in species composition, though highly valuable, do not give a complete representation of ongoing changes in Arctic megafauna biodiversity.

Investigations on climate change impacts on Arctic marine ecosystem functioning are equally scarce.

Griffith et al. [START_REF] Griffith | Ecological resilience of Arctic marine food webs to climate change[END_REF] modelled the arrival of Atlantic species (such as capelin Mallotus villosus) and showed they may increase the resilience of marine fjord systems, with maintained food web structure.

Further, Frainer et al [START_REF] Frainer | Climate-driven changes in functional biogeography of Arctic marine fish communities[END_REF] based their work on fish functional biogeography to address how ecosystem functioning may be affected by climate change in the Barents Sea. Their results also support an ongoing borealisation of Arctic marine ecosystems with large boreal species replacing small Arctic ones, likely affecting biomass production. Other analyses of functional redundancy [START_REF] Aune | Functional roles and redundancy of demersal Barents Sea fish: ecological implications of environmental change[END_REF] or specific species interactions [START_REF] Dey | Increasing nest predation will be insufficient to maintain polar bear body condition in the face of sea ice loss[END_REF][START_REF] Breed | Sustained disruption of narwhal habitat use and behavior in the presence of Arctic killer whales[END_REF] have also been performed, to explore changes in ecosystem functioning due to climate change. These studies confirm that ongoing climate change has the potential to affect Arctic marine ecosystem functioning, even though the exact consequences remain extremely difficult to apprehend.

The way forward

Rigorously assessing climate change impacts on marine megafauna at the scale of the Arctic is a formidable task (Figure 3). Ecologists thereby face the combined challenges of an immensely vast terrain, of drastic weather conditions and staggering operational costs [START_REF] Mallory | Financial costs of conducting science in the Arctic: examples from seabird research[END_REF]. In addition, the current socio-economic crisis questions the environmental footprint of research operations and reduces available funding. Finally, international tensions prevent collaboration between Russian scientists and the rest of the world. This considerably slows AMM data acquisition in least-known areas.

Despite these hurdles, and thanks to the dedication of passionate individuals within Arctic communities and the international scientific community, research on marine megafauna is progressing. Three powerful leverages permit such advances, now and in the near future.

(1) Modern technologies allow remote, large-scale data collection on previously totally unknown aspects of AMM ecology. This starts with satellite remote-sensing of multiple biotic and abiotic parameters all across the Arctic, which nonetheless only assesses conditions at the sea surface. In situ, aerial and underwater autonomous vehicles (e.g. drones) automatically survey areas ranging from one, to millions of cubic meters of ocean [START_REF] Gray | Drones address an observational blind spot for biological oceanography[END_REF]. They provide fine-scale information on environmental conditions, notably on the spatio-temporal abundance of potential prey for marine megafauna (e.g. zooplankton and small pelagic fish aggregations). In addition, biologging devices attached to animals opened worlds of knowledge on their spatial ecology and their energetics in a changing ocean [START_REF] Davidson | Ecological insights from three decades of animal movement tracking across a changing Arctic[END_REF],

and combined analyses provide information on contaminant levels in animals and their environment [START_REF] Sonne | Emerging contaminants and biological effects in Arctic wildlife[END_REF]. Finally, further rapidly emerging techniques such as DNA metabarcoding are transforming the field of population ecology [START_REF] Bohmann | Environmental DNA for wildlife biology and biodiversity monitoring[END_REF].

(2) Beyond these technological revolutions in data acquisition, marine megafauna ecology has now entered the realm of big data science: Information technology allows the design of completely new frameworks for acquiring, storing, sharing, analyzing, visualizing, and publicizing data [START_REF] Grémillet | Big data approaches to the spatial ecology and conservation of marine megafauna[END_REF]. Big data approaches are notably based upon the use of artificial intelligence and deep learning scheme which drastically reduce analysis duration and costs [START_REF] Saleh | Computer vision and deep learning for fish classification in underwater habitats: A survey[END_REF][START_REF] Hayes | Drones and deep learning produce accurate and efficient monitoring of large-scale seabird colonies[END_REF], and online platforms greatly enhance data sharing [START_REF] Davidson | Ecological insights from three decades of animal movement tracking across a changing Arctic[END_REF].

(3) All above-mentioned approaches may be complemented with citizen-science initiatives [START_REF] Brown | The potential for citizen science to produce reliable and useful information in ecology[END_REF][START_REF] Peacock | Linking co-monitoring to co-management: bringing together local, traditional, and scientific knowledge in a wildlife status assessment framework[END_REF].

Those may allow additional data collection within scientific blind spots, notably species distribution and abundance, and assist in long-term monitoring when such activities become impossible for conventional research teams, as during the recent COVID19 pandemic. Participatory science programs may also contribute to the empowerment of local communities, in many areas of the Arctic where the predominance of non-Indigenous scientists may be perceived as post-colonial. These research targets, which aim at understanding global change impacts on Arctic marine megafauna while better involving local communities, are highly coherent with the objectives of the Arctic Marine Biodiversity Monitoring Plan of the Arctic Council [START_REF] Gill | Arctic Marine Biodiversity Monitoring Plan[END_REF]. This initiative should use scientific evidence to improve biological conservation in the Arctic, but the political will and international collaboration necessary to transform research into action still seems in its infancy [START_REF] Barry | How does the Arctic Council support conservation of Arctic biodiversity?[END_REF].

Concluding Remarks: Key research gaps and questions on Arctic marine megafauna

• With a few exceptions, it is not possible to conclude on recent trends in AMM populations and the inter-species and spatial variations in these trends. Basic data about population size are still lacking for most species and regions. From these knowledge gaps, as well as following two decades of interactions with the Arctic research community and its many panarctic stakeholders, in particular the Arctic Council and its working groups, we identified ten research avenues (see outstanding questions). Those are particularly far-ranging, and aim at presenting a general framework beyond our review of recent knowledge. They start with interrogations about individual and populational responses of AMM to climate change, subsequently leading to wider considerations at the interface between the fate of AMM and that of Arctic peoples.

Glossary • Arctic amplification

The arctic amplification refers to faster warming in the Arctic as compared to the rest of the globe (nearly four times faster during 1979-2021). This amplification can be explained by several factors, among which changes in albedo due to decreasing sea-ice, and ocean heat transport.

• Borealisation

The borealisation of the Arctic refers to the ongoing expansion of so called "boreal" (i.e. north temperate) species into arctic biomes. In the marine environment, this process may be driven by the advection of warmer and saltier waters from the Atlantic or Pacific oceans into the polar basins. These phenomena are called "Atlantification", or "Pacification", of the Arctic.

• Gill-oxygen limitation theory

According to the Gill-Oxygen Limitation Theory, the oxygen carrying-capacity of water is limited by its temperature, salinity, and pressure. Colder water therefore holds more dissolved oxygen than warmer water. As water temperature increases or salinity decreases, the oxygen-carrying capacity of the water decreases, making it more difficult for aquatic organisms to extract oxygen. In fishes, growth and maximum sizes are consequently limited by water oxygen carrying-capacity and the size of the gills.

• Cryosphere

The cryosphere is defined as the part of Earth's surface layer consisting of frozen water in the form of snow, permafrost, glaciers and sea-ice.

• Tipping point

In the context of climate change, tipping points refer to critical thresholds in climate conditions, above which abrupt and potentially irreversible changes in ecosystem structure and dynamics occur.

• Shannon diversity

Shannon diversity is a biodiversity measure that takes into account the number of different species present in a given ecosystem and their relative abundance.

• Functional biogeography

The field of functional biogeography combines knowledge on species distribution with information on species' functional traits, to understand how large-scale distributional changes may affect ecosystem functioning.

• Biologging

Biologging refers to the study of living organisms via the use of data-recording devices. In animal ecology, biologging technologies are generally used to gather information on animal behavior (e.g. 3D movement) or physiology (e.g. heart rate).

• DNA metabarcoding DNA metabarcoding is a molecular technique that allows identifying the species present in a sample, (e.g. soil, water) by analyzing all DNA sequences present in this sample and comparing them to reference databases.

• Deep learning

Deep learning is a branch of artificial intelligence (AI) that uses algorithms to enable computer systems to learn and improve from experience without being explicitly programmed. In ecology, deep learning is used to automatically detect specific features in large and/or complex data (e.g. to identify or count individuals, classify behaviors).

• Ecotoxicology

Ecotoxicology is the study of the toxic chemicals present in living organisms. It aims at understanding the mechanisms of toxicity and assessing consequences on organism physiology, behavior or demography. For instance, persistent organic pollutants (POPs) and per-and polyfluoroalkyl substances (PFAS) are key contaminants in Arctic wildlife with known detrimental consequences. Table 1. Main topics addressed in studies linking marine megafauna and climate change. Each cell of the table gives the % of studies dealing with each topic. The sum for each guild is usually >100% as a given study may address several topics. "Community" refers to studies dealing with ecosystem or community structure, "Habitat use" to studies dealing with spatial distribution, habitat use or movement, "Population dynamics" to studies dealing with population trajectory, abundance or age structure, "Demography" to studies dealing with vital rates (survival, reproduction) or phenology, and "Individual state" to studies dealing with body condition, physiology, energetics or pollutants. The column "Other" corresponds to a variety of themes and includes reviews or opinion articles, as well as studies based on Indigenous ecological knowledge. guild is indicated under the guild icons (note that a given study may concern several guilds, so that the total number of studies here does not correspond to the total number of studies reported in our review).

Community

•

  Individual variations in responses to climate change, their drivers (e.g. age, physiological status, experience) and their implications to buffer climate change impacts also remain largely unknown. Such individual variations are, however, crucial to assess populational responses to environmental changes [100]. • The shape of the relationships (linear/non-linear) between AMM physiological, behavioural or demographic traits and the direct and indirect (e.g. pollutants and pathogens, shifting human activities) consequences of climate change are critically understudied. This prevents any reliable predictions about the fate of AMM in response to future climate change scenarios.

Figure 1 :

 1 Figure 1: Examples of Arctic marine megafauna. Top: Arctic tern Sterna paradisaea (credit S. Descamps), Atlantic cod Gadus morhua (W. T. Fiege), Walrus Odobenus rosmarus (D. Grémillet). Centre: Gonatus fabricii, the Boreo-atlantic Armhook Squid (www.descna.com), Common eider Somateria mollissima (D. Grémillet). White-beaked dolphin Lagenorhynchus albirostris (foreground) and fin whale Balaenoptera physalus (background) (S. Descamps). Bottom: Bearded seal Erignathus barbatus (S. Descamps), Polar cod Boreogadus saida (P. Leopold), Polar bear Ursus maritimus (M. Andersen).

Figure 2 :

 2 Figure 2: Distribution of recent Arctic marine megafauna studies (see methods and Suppl. 1 for definitions and time frame). Colours correspond to the different megafauna groups as pictured below the map, and the proportion of each colour per pie is indicative of the number of studies per megafauna group and area. Pies are sized relative to the total number of megafauna studies per area (see methods). The vast majority of the Russian studies took place in the Barents sea. The number on each pie represents the number of studies dealing with each guild. The total number of studies per

Figure 3 :

 3 Figure 3: The way forward -Outline of future research work, necessary to assess climate change impacts on Arctic marine megafauna. Climate change affects individual vital rates through changes in physiology, behaviour or even morphology. These effects can be mediated by changes in habitats (e.g. connectivity) or trophic relationships (e.g. dietary changes through shifts in prey availability, changes in inter-species competition). The integration of complementary methods based on new technologies, new statistical methods, as well as citizen science are needed to understand ongoing changes in megafauna populations and marine ecosystems.

Acknowledgements

DG is funded by the French National Center for Scientific Research (CNRS) and the French Polar Institute IPEV (through the ADACLIM program IPEV 388, led by David Grémillet and Jérôme Fort), and the European Horizon 2020 Program EcoScope project under the grand agreement number 101000302. SD is funded by the program SEAPOP (http://www.seapop.no/en/), the program MOSJ (http://www.mosj.no/en/) and the project FACE-IT (The Future of Arctic Coastal Ecosystems -Identifying Transitions in Fjord Systems and Adjacent Coastal Areas) funded by the European Union's Horizon 2020 research and innovation program under grant agreement number 869154. We thank Manon Amiguet, Maria Gavrilo and Joanna Sulich for bibliographic and illustrative assistance.

Author contributions

DG and SD conceived the study, performed the bibliographic analysis and wrote the paper.

Declaration of interests

No interests are declared

Supplementary information

Supplementary information associated with this article can be found, in the online version, at ##


Krzysztof K Dudek

Julio Andrés

Iglesias Martinez

Gwenn Ulliac

Muamer Kadic
email: muamer.kadic@femto-st.fr

J A Iglesias Martínez

Micro-Scale Auxetic Hierarchical Mechanical Metamaterials for Shape Morphing

Keywords: mechanical metamaterials, hierarchical metamaterials, auxetic, shape morphing

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Shape morphing [1,2,3,4,5,6,7,8], utilising the topological or space time properties [9,10,11] and the ability to control mechanical properties [START_REF] Miltion | The Theory of Composites[END_REF] of functional materials [START_REF] Milton | [END_REF]14,15,16,17,18,19,20,21,22,23] remain some of the main challenges in materials science. At the same time, the possibility of constructing materials possessing such properties is in high demand as it may lead to the design of structures superior to currently known biomedical and other devices used in various industries. In addition, the task of designing and manufacturing such materials becomes even more difficult at small scales such as the microscale that are crucial from the point of view of many new types of applications, e.g. flexible electronics [24,25] and specialized medical equipment [26,27]. However, despite the level of difficulty, over the years, it has been possible to note an emergence of promising directions of studies that address the possibility of observing these effects. In fact, one of the most interesting directions of studies related to this topic are mechanical metamaterials [28,29,30,3,31,32], i.e. structures that can exhibit counterintuitive mechanical behavior based primarily on their design. Mechanical metamaterials are known for their ability to exhibit counterintuitive mechanical properties such as auxetic behavior [33,34,35,36,37,38,39,40], negative stiffness [2,41] and negative compressibility [42,43] that are essential in many industries. However, in a vast majority of cases, standard mechanical metamaterial proved insufficient while searching for structures capable of exhibiting versatile deformation patterns. This in turn led to intensive studies devoted to hierarchical mechanical metamaterials [44,45,[START_REF] Cho | Proc. Natl. Acad. Sci[END_REF], i.e. structures composed of elements having their own geometry that normally can deform irrespective of the rest of the system. Despite the large popularity of hierarchical mechanical metamaterials, it is a relatively new thread in the field of mechanical metamaterials. It seems that some of the first studies devoted to this topic were published by Gatt et al. [45] and Cho et al. [START_REF] Cho | Proc. Natl. Acad. Sci[END_REF] where the famous hierarchical rotating square system was proposed. In the following years, it was demonstrated [45,[START_REF] Cho | Proc. Natl. Acad. Sci[END_REF][START_REF] Tang | [END_REF]48,49] that this structure can exhibit tunable auxetic behavior where the extent of auxeticity depends on the relative rate of the deformation of hierarchical levels constituting the system. Recently, it was also reported that the design of the hierarchical square system can be modified in order to change its characteristic [50,51,4,52,53]. Similar studies based primarily on the extent of the observed auxeticity were also conducted for structures based on rotating rectangles [54] as well as re-entrant and other honeycombs [54,55,56,57,58].

Even though the concept of hierarchical mechanical metamaterials proved to be very popular and resulted in numerous publications, it is possible to note that the hierarchical structures proposed up to date normally share several limitations. First, most of the known hierarchical mechanical metamaterials are designed in a way where their different hierarchical levels correspond either to the same deformation mechanism or to structures having very similar Poisson's ratio. Hence, the possible range of the resultant auxetic behavior is relatively small and may prove to be insufficient in the case of applications where functional materials must significantly change their mechanical response. Another limitation of the already reported hierarchical metamaterials is the fact that in a vast majority of cases, their ability to exhibit the tunable mechanical behavior was only demonstrated for macroscopic prototypes. On the other hand, there are numerous types of applications (e.g. biomedical devices and flexible electronics) where similar materials with the ability to exhibit programmable auxetic behavior would be of great importance at a much lower scale with the emphasis on the mico-scale. Finally, almost all of the reported hierarchical metamaterials are two-dimensional systems which lowers their applicability. Thus, the possibility of designing three-dimensional hierarchical metamaterials [59,60,61] capable of exhibiting programmable control over mechanical properties in multiple directions would be of great significance.

We propose novel 2D and 3D micro-scale hierarchical mechanical metamaterials. In addition, by means of experiments supported by Finite Element Method (FEM) simulations, it is demonstrated that the considered structure is capable of exhibiting controllable auxetic behavior upon changing the thickness of hinges connecting its structural elements. In this regard, thanks to the very novel design resulting in the mutually-competing substructures being present within the system, it is showcased that the analyzed model may exhibit a very broad range of the auxetic behavior which in turn increases its applicability and the appeal for researchers in the field of materials science. Finally, it is shown that the analyzed mechanical metamaterial may exhibit a predefined shape morphing. In other words, it is shown that thanks to its hierarchical design, the considered mechanical metamaterial can change its shape in order to match the shape of another object upon being subjected to an external stimulus.

Results and discussion

In this work, novel 2D and 3D hierarchical mechanical metamaterials are designed in order to assess their ability to exhibit a wide range of auxetic behavior as well as shape morphing. As shown in Fig. 1(a), the 2D version of the considered system is a two-level hierarchical structure. However, unlike in the case of a vast majority of the already known hierarchical mechanical metamaterials, the deformation mechanisms corresponding to each of the hierarchical levels of the system are very different. More specifically, as shown in Fig. 1(b), the structure selected to form level 0 of the system corresponds to the nonauxetic rotating triangles structure with its unit-cell having a shape of a rectangle. This geometry was first proposed by Milton et al. [START_REF] Milton | [END_REF] where it was used in order to construct mechanical expanders capable of significantly changing their dimensions during the deformation process. A few years later, Dudek et al. [62] demonstrated that this specific mechanical metamaterial never exhibits auxetic behavior in the axial directions, and instead it can be characterized by the negative linear compressibility irrespective of the geometric parameters used to define it. On the other hand, the deformation mechanism associated with level 1 of the structure corresponds to the rotating square [63] / rectangle [64] system proposed initially by Grima et al. with the rotating square-like geometries being famous for their ability to exhibit auxetic behavior. In view of this, the deformation of the hierarchical system considered in this work corresponds to the interplay between the two hierarchical levels associated with a positive (level 0) and negative (level 1) Poisson's ratio respectively (this interplay is referred to as mutually-competing substructures). Thus, depending on the extent of the mechanical deformation of the two hierarchical levels, it is expected that the Poisson's ratio of the entire structure can be significantly modified. At this point, it should be mentioned that even though in this work a specific configuration of rotating triangles is considered, in the literature, there are also other types of triangle-based geometries [65,66,67,68] that in general may exhibit other forms of the unusual mechanical behavior.

In terms of the geometry, the two-dimensional analog of the analyzed structure corresponds to the two- level hierarchical structure where level 0 is composed of structural elements closely resembling isosceles triangles (see Fig. 1(a)). On the other hand, level 1 structural elements correspond to rectangle-like motifs that for the specific case of θ 0 = 90 • assume a shape of a square. In the case of the provided example (see Fig. 1(a)), level 1 structural elements are composed of 2 × 2 level 0 units. However, in general, within the frame of the considered model, level 1 blocks can be constructed by means of a different number of 2N x × 2N y level 0 elements (e.g. 4 × 4) assuming that they form a rectangle-like lattice and are connected to each other as shown in Fig. 1. Furthermore, one should note that even though the structure portrayed in Fig. 1(d) is being referred to as being two-dimensional, in reality, it has a non-zero out-of-plane thickness that corresponds to the variable l s . In addition, it should be emphasized that the unit-cell of the 3D version of the considered hierarchical mechanical metamaterial is very similar to its 2D equivalent. As shown in Fig. 1(c), walls of the unit-cell of the 3D system are almost identical to the 2D unit-cell with the addition of solid blocks having dimensions l × l × h that help to connect adjacent walls.

In this work, to analyze the mechanical behavior of the considered hierarchical structure, the entire system is being compressed along the z-axis (see Fig. 1) with its bottommost elements being fixed in space. Furthermore, to assess its ability to exhibit a versatile mechanical behavior with a broad range of the exhibited Poisson's ratio, several different structures are investigated with each of them corresponding to a different thickness of hinges connecting structural elements.

2D hierarchical metamaterials -control over mechanical properties

In order to assess the ability of the considered model to exhibit a tunable Poisson's ratio, three different quasi-2D hierarchical structures (see Fig. 2) are considered with the main difference between them being the thickness of hinges connecting level 0 and level 1 elements. As shown in Fig. 2(a), the first of the analyzed structures (case 1) corresponds to the hierarchical system where level 1 building blocks assume a shape of a square since θ 0 = 90 • . Based on the pictures taken during the experiment, one can note that during the deformation process corresponding to the compression along the z-axis (see Supplementary Video 1), the considered part of the structure, i.e. the central topmost unit-cell, acts as a typical rotating square system. In other words, level 1 structural blocks retain their shape throughout the deformation and rotate with respect to each other which leads to a change in the angle θ 1 . As presented in Fig. 2(d), this deformation mechanism results in the highly negative and almost isotropic Poisson's ratio close to -1, i.e. the extent of auxeticity characteristic for the rotating square system. The specific behavior of the structure referred to in this work as case 1 stems from the fact that hinges connecting level 0 elements are thicker than hinges connecting level 1 blocks (d 1 ≈ 9.3 µm, d 2 ≈ 6.1 µm). At the same time, all hinges are thin enough so that all of the unit-cells deform primarily through hinging between their structural elements. As a result, it is much easier for the level 1 blocks to rotate with respect to each other than is the case for level 0 elements where the resistance to the rotational motion is significantly larger. Hence, during the deformation process, θ 1 changes to a large extent while θ 0 approximately retains its value (see Fig. 2(e)). At this point, it should be also noted that even though the initial configuration of the system allows for a very large extent of the mechanical deformation, it does not correspond to the fully-open configuration, where θ 0 = 90 • and θ 1 = 90 • . This stems from the fact that for such system, it would be difficult to predict the direction of rotation of structural elements since it would depend on the polymer's composition and printing imperfections (see Supplementary Information).

According to Fig. 2(b), the hierarchical structure referred to as case 2 exhibits a very different behavior than was observed for case 1. More specifically, during the deformation process (see Supplementary Video 2), level 1 blocks having a rectangular shape become significantly flattened in one dimension with the orthogonal dimension being slightly increased (i.e. level 1 blocks exhibit the negative linear compressibility behavior characteristic for the non-auxetic rotating triangle system). Furthermore, the change in the shape of the level 1 blocks is accompanied by the mutual rotation of level 0 elements to a significant extent (see Fig. 2(e)). As shown in Fig. 2(d), this change in the behavior of the hierarchical structure significantly changes the Poisson's ratio that could be observed for the system referred to as case 1. In fact, the Poisson's ratio assumes much less negative values throughout the entire deformation process that reach the level of -0.38 for the experiment. This drastic change in the behavior of the analyzed system originates from the fact that hinges connecting level 0 elements are significantly thinner than hinges corresponding to level 1 of the system (d 1 ≈ 2.7 µm, d 2 ≈ 10.0 µm). As a result, despite a large number of hinges associated with level 0 of the system (16 within the unit-cell), the overall resistance of level 0 elements to the rotational motion is not much different than in the case of level 1. Thus, both hierarchical levels could deform to a significant extent which process is reflected by a large concurrent change in angles θ 0 and θ 1 (see Fig. 2(e)). At this point, it should also be noted that for cases 1 and 2, a part of the bottommost row of unit-cells is not captured on the provided pictures. This stems from the fact that the lenses used in the experiment (see Methods section) were selected in a way ensuring a high quality of this part of the picture that was taken into account while assessing mechanical properties of the system.

The last of the analyzed 2D structures is referred to as case 3 (see Fig. 2(c)) and corresponds to a very similar system to the one referred to as case 2. In fact, all of the initial angles, as well as thicknesses of hinges, are almost the same in terms of the aspect ratio for both structures. The only significant difference between them corresponds to the number of level 0 elements constituting level 1 blocks, i.e. for case 3, level 1 blocks consist of 16 level 0 elements instead of 4. As shown in Fig. 2(c), this small difference in the design of the structure results in a very different behavior of the hierarchical system during the deformation process (see Supplementary Video 3). More specifically, level 1 blocks do not get visibly flattened with respect to the initial configuration as was the case for case 2. Instead, they approximately retain their shape throughout the entire deformation process. According to Fig. 2(d), the change in the number of level 0 units also significantly affects mechanical properties of the system resulting in the greater extent of the auxeticity. This change in the behavior between case 2 and case 3 can be explained upon having a closer look at the number of hinges present within the system. More specifically, upon increasing the number of level 0 units constituting level 1 blocks, the number of hinges connecting level 0 elements is also significantly increased. At the same time, the number of hinges connecting level 1 blocks remains the same. Thus, upon comparing cases 2 and 3, it is much more difficult to rotate level 0 elements corresponding to case 3. As a result, the deformation of this structure corresponds primarily to the change in the value of θ 1 as opposed to the variation in θ 0 (see Fig. 2(e)).

At this point, it is also worth noting that all of the structures considered in this study deform primarily through the rotation of their structural elements and not their flexing. More specifically, as shown in Fig. 2, according to the von Mises stress distribution corresponding to the FEA simulations, the main accumulation of the stress can be observed in the vicinity of hinges connecting level 1 blocks. On the other hand, in the remaining parts of the structure, it assumes much lower values not exceeding 50 MPa. Since Young's modulus of the polymer used to manufacture the samples is equal to 4GPa (see Methods section), this means that the local stresses do not result in the change of the shape of structural elements. Instead, as shown on the provided pictures, structural elements approximately retain their shape throughout the entire deformation process. Furthermore, it should be noted that even though in this work we focus on the longitudinal compression of the analyzed structures, in general, one can also compress them from other directions including the transverse direction. As shown in the Supplementary Information, the compression of the considered systems in the transverse direction may affect their mechanical properties. Nevertheless, not all of them are affected by a change in the direction of compression to the same extent. More specifically, the behavior of the case 1 system seems to be only marginally affected by the direction of compression. On the other hand, especially at low strains, the Poisson's ratio corresponding to the case 2 structure is visibly altered.

3D hierarchical system

As mentioned in the Introduction, almost all of the known hierarchical mechanical metamaterials are two-dimensional structures which limits their applicability as they can only exhibit their properties in one plane. Thus, it is essential to identify structures that could also exhibit a controllable mechanical response in the case of 3D systems. In fact, one of the first such structures is the hierarchical metamaterial proposed in this work as its ability to exhibit a very broad range of the auxetic behavior is not limited to 2D structures and can be conveniently extended to 3D models. To demonstrate it, we selected two very similar structures referred to as type A and type B. These structures have unit-cells defined as shown in Fig. 1(c). In addition, the aspect ratios of hinges and other geometric dimensions corresponding to type A and type B mechanical metamaterials are the same as in the case of the quasi-2D structures named case 1 and case 2 respectively. Hence, based on the already generated results, one should expect the type A structure to be able to exhibit a more auxetic behavior than the type B system. It should be also noted that type A and type B structures look almost identical with the main difference between them being the thickness of hinges. Thus, as an example, a picture of the type A system is shown in Fig. 3(b).

As shown in Fig. 3(a-b), it is indeed possible to construct the microscopic 3D equivalent of the quasi-2D models considered in this work. In addition, upon designing the system composed of unit-cells having the same thickness of hinges as the 2D structure called case 1, it is possible to observe a very strong auxetic response similarly to what was observed for its 2D counterpart (see Fig. 3(a) and Supplementary Video 4). Of course, the extent of the auxeticity in the xz and zy planes is smaller than for the 2D structure and for selected parameters, it maintains the value in the vicinity of -0.4 throughout the entire deformation process (see Fig. 3(c)). This change in the magnitude of the Poisson's ratio originates from the presence of additional blocks that increase the lateral dimension of the unit-cell in comparison to the case 1 2D structure. Furthermore, upon comparing results corresponding to type A and type B, one can note that in the latter case, the Poisson's ratio assumes significantly less negative values. This in turn is an indication of a possibility to achieve a tunable auxetic behavior similarly to what was the case for 2D systems. It should be also emphasized that the range of negative Poisson's ratio for 3D systems could be further increased if faces of 3D unit-cells were composed of several adjacent structural motifs similar to the geometry presented in Fig. 1(a). Nevertheless, at the microscale, it would also result in a more challenging manufacturing process.

Shape morphing

In addition to the possibility of controlling the magnitude of the Poisson's ratio exhibited by the analyzed system, another very interesting direction of studies corresponds to the ability of the hierarchical structure to change its shape during the deformation process. In fact, in Fig. 2, it was shown that depending on the thicknesses of hinges, the entire unit-cell may assume different shapes. Thus, in theory, if one would construct the composite mechanical metamaterial consisting of unit-cells associated with multiple different sets of geometric parameters, then it would be possible to obtain the hierarchical structure with the local change in shape as well as mechanical properties. To demonstrate it, one such example is considered in this study.

As shown in Fig. 4, the hierarchical mechanical metamaterial composed of 4 × 3 × 4 case 1 unit-cells and 4 × 3 × 2 case 2 unit-cells (see Fig. 4(a-b)) is considered in order to analyse its ability to change shape in a controllable manner. More specifically, the objective is to change the shape of the initial cuboid-like structure to match the shape of the reference object in the form of the mug presented in Fig. 4(d) (see Figure 4: Shape morphing of the considered hierarchical mechanical metamaterials aimed towards the possibility of replicating the shape of the reference object. a) A graphical representation of unit-cells corresponding to case 1 and case 2 that were used in the design of the structure that is analyzed from the point of view of its ability to change shape in a predefined manner. b) Picture of the prototype used in the experiment together with the zoomed-in images of types of structures used in the design of the resultant composite material. c) The experiment corresponding to the compression of the considered structure that results in the change in its shape. d) The real-life mug used as an inspiration for the shape that the analyzed hierarchical system is supposed to assume upon being deformed. Supplementary Video 5). According to Fig. 4(c), upon compressing the system, its shape changes drastically with the bottom part composed of case 1 cells shrinking significantly in the lateral dimension. This effect originates from the strong auxetic behavior that is characteristic for case 1 unit-cells (see Fig. 2). On the other hand, even though the top part of the hierarchical system consisting of case 2 unit-cells also shrinks in the lateral dimension, the extent of such shrinkage is much smaller than is observed for the bottom part of the structure. This change in the behavior can be explained by the much smaller extent of the auxeticity corresponding to the unit-cell referred to as case 2. Thus, overall, Fig. 4(c-d) shows that by designing the hierarchical structure considered in this work in the specific compositelike manner, the entire system may undergo a shape morphing into a much more complex configuration. Of course, the considered example is relatively simple but it proves that in general, one can use the concept presented in this work in order to observe very complex shape morphing at the micro-scale. At this point, it is also worth to mention that in the case of the specific design considered in this study, the two types of materials were connected by means of an additional thin plate that was constraining topmost case 1 cells and bottommost case 2 cells. As a result, at the end of the deformation process, one can observe a big difference in the horizontal dimension of the middle and the top part of the case 1 structure. In fact, the same effect could be achieved if one was to replace the plate with one additional row of very stiff unit-cells. Of course, without the additional plate, a similar behavior is still expected to be observed due to a large difference in the Poisson's ratio between the two types of structures. Nevertheless, such system would be more difficult to manufacture and the change in the horizontal dimension of the middle and top part of the case 1 structure would be expected to be smaller.

The results reported in this study clearly show that the considered hierarchical structure can be constructed at the microscale and that its mechanical properties with the emphasis on the exhibited Poisson's ratio may be controlled and significantly modified depending on the small change in the design parameters. It should be also mentioned that in theory, such control over the extent of the exhibited auxeticity could be achieved in an active manner, i.e. without the need of reconstructing the system. In this regard, it should be noted that in the case of this work, the behavior of the hierarchical structures is considered for the quasi-static compression where the moment of inertia of structural elements does not affect the results. However, for fast deformations, the rate at which the structure is being compressed could become another factor that allows to determine whether both hierarchical levels are deforming to a large extent. Of course, for this effect to be observed, hinges would have to be appropriately thin with respect to the considered deformation rates (see Supplementary Information). In addition, the active control over the behavior of the structure could be achieved through the use of external stimulus such as a change in temperature assuming the use of materials corresponding to the high thermal expansion coefficient while designing the hinges.

In view of all of the above, it is important to emphasize the fact that the results of this work may prove to be of great significance in the case of numerous applications. First of all, the ability of the considered hierarchical mechanical metamaterial to exhibit a very broad range of the auxetic behavior may lead to the design of highly efficient protective equipment (especially for the macroscopic version of the considered concept). In fact, as shown in the Supplementary Information, depending on the type of the analyzed structure, the specific energy absorption of the system may be significantly adjusted with its estimated values ranging between 133.2 J kg -1 and 386.6 J kg -1 . Hence, the considered tunable properties are also interesting from the perspective of impact absorption devices. In addition, the concept proposed in this work may be used in the design of tunable vibration dampers utilizing local vibration isolation. This stems from the fact that as shown in the Supplementary Information, the stiffness of the system can be adjusted depending amongst others on the thickness of its hinges. This in turn is a commonly desired feature related to vibration damping devices. In addition, the considered concept is particularly interesting since the model proposed in this work is not solely limited to two-dimensional structures where the devices making use of the provided results could only exhibit their properties in a single plane. Furthermore, the ability of the analyzed system to exhibit shape morphing may significantly increase the efficiency of multiple medical devices including stents by providing localized support for specific parts of the blood vessel that need additional reinforcement. Finally, the shape morphing ability could also be utilized in the design of flexible electronics and equipment used on spacecraft that could adjust its shape upon being deployed from the initially confined configuration that does not waste space onboard prior to being used.

Conclusion

In this work, it is shown that the novel microscopic hierarchical mechanical metamaterial can exhibit a wide range of the auxetic behavior depending solely on the change in the thickness of hinges connecting its structural elements. It is also demonstrated that the considered concept can be observed both for the quasi-2D and 3D version of the structure which enables the system to exhibit a desired mechanical response upon being subjected to a deformation induced from different directions. Furthermore, it is presented that the considered structure can exhibit shape morphing allowing it to match the shape of the reference object upon being deformed. It should be noted that different aspects of these results offer a new perspective with regards to the capabilities of hierarchical mechanical metamaterials and are likely to pave the way for other researchers to design similar microscopic functional materials that will further enhance their properties. Finally, it should be emphasized that the presented results may be utilized in numerous applications. More specifically, the ability of considered hierarchical metamaterials to exhibit a broad range of the auxetic behavior could be important in the design of the protective equipment and vibration dampers. On the other hand, the ability of the considered structure to exhibit shape morphing is very promising for example from the point of view of medical devices capable of locally adjusting their properties as well as flexible electronics.

Methods

Fabrication

The micro-scale samples investigated in this work were fabricated by polymerizing individual unit-cells assuming a small overlap between the adjacent cells. Such overlap was introduced to ensure that the adjacent unit-cells would be connected to each other to form larger composites analyzed in this study. To this aim, the commercial 3D printer (Photonic Professional GT+, Nanoscribe GmbH) operating based on the two-photon lithography method was used. The photoresin selected to produce the analyzed mechanical metamaterials was the commercial negative tone IP-S resin (Nanoscribe GmbH) that is customized to work well with the Nanoscribe 3D printer. Furthermore, the slicing and hatching distances associated with the samples were set to be equal to 1 µm and 0.5 µm respectively. A drop of IP-S resin was deposited on a ITO-coated soda lime glass substrate with dimensions 25 × 25 × 0.7 mm 3 and photopolymerized with a femtosecond laser operating at λ = 780 nm and a 25X-objective. After printing, the sample was developed for 25 min in Propylene glycol methyl ether acetate (PGMEA) solution to remove the unexposed photoresist and rinsed for 3 min in Isopropyl alcohol (IPA) to clear the developer. A Laser Power of 90 and a Galvanometric scan speed of 100 mm s -1 were used for the whole fabrication process. Finally, to improve the adhesion properties of IP-S resin and so to ensure a better connection of the printed sample with the substrate, ITO-coated substrates are pre-treated with oxygen plasma (Corial 200R, RIE etch system) for 5 min. As a result, the bottom part of the samples was constrained from the movement during the deformation process.

Testing

To test the mechanical properties of the analyzed samples, they were uniaxially compressed at the constant rate of 1 µm/s. The intendter used to induce the compression had a circular polished surface corresponding to the diameter approximately equal to 3 mm. Each sample was placed on top of the leveling table in order to ensure its correct orientation during the experiment. In addition, to ensure the better stabilization, the entire set-up was also placed on top of the optical table. To record the deformation process, an optical camera equipped with a lense corresponding to the 20x and 10x magnification factor was used. It is also worth to mention that prior to the testing, in order to have high-resolution pictures showing the structural details of the printed samples, the SEM microscope was used (Apreo S -Thermofisher). Finally, it should be emphasized that the experiments were continued until the point when hinges connecting adjacent structural elements were breaking at large strains. In addition, to assess whether a given breaking hinge was significantly affecting the deformation pattern, the force vs displacement relationship was analyzed throughout the experiment (see Supplementary Information). It should be also emphasized that if one was to construct the system with more durable hinges, then considered structures could be deformed to an even larger extent than is the case in this study. Furthermore, it should be mentioned that the experimental data presented in Fig. 2 were obtained based on pictures taken during the experiment by means of the optical camera. To this aim, positions of characteristic vertices (e.g. edges of hinges connecting level 0 and level 1 blocks) were extracted from respective pictures and utilized to calculate the desired geometric parameters by means of standard mathematical operations.

FEA simulations

To ensure the validity of the experimental results, the mechanical behavior of the analyzed hierarchical metamaterials was analyzed by means of the FEA simulations. To do this, COMSOL Multiphysics software was used (see Supplementary Information to find governing equations corresponding to the considered type of FEM simulations). Since the behavior of the structure was investigated solely from the point of view of its deformation, only the Structural Mechanics module was implemented. In addition, to minimize the computation time, the geometries of the quasi-2D experimental prototypes analyzed in this study were converted into regular 2D structures. Furthermore, to replicate the behavior of the experimental prototypes, the surfaces at the bottom of the structure were fixed in space. Thus, as the samples were compressed, the bottom part of the system could not move. Such deformation process associated with the uniaxial compression of the system was induced through the use of the external indenter pushing the topmost part of the structure downwards. Finally, it should be mentioned that the material corresponding to the analyzed samples was assumed to be isotropic and its properties were set to be the following: ν = 0.4, E = 4 GPa. It should be noted that for the considered structures, the selection of the model of elasticity does not significantly affect the mechanical behavior of the system since it depends primarily on its design (see Supplementary Information). Furthermore, it should be emphasized that while the considered geometry is nonlinear, the conducted FEM simulations were linear. This stems from the fact that the considered structures are deforming primarily through hinging of structural elements that are retaining their shape throughout the deformation. Thus, the plasticity of a material is not significant from the point of view of the deformation of considered structures and internal stresses are relatively small in comparison to the Young's modulus of the polymer. At this point, even though the following does not belong to the scope of this work, it should be also mentioned that deriving dynamics-related properties would be very difficult in the general case for this work since they would always depend on the material's non-linear properties [69].

Figure 1 :

 1 Figure 1: The design of 2D and 3D hierarchical structure considered in this work. a) A cross-section of the unit-cell of the considered model having level 1 structural elements composed of 4 (2 × 2) level 0 units. b) Conceptual deformation of substructures corresponding to level 0 and level 1 of the analyzed system. c) The unit-cell of the 3D model considered in this study. d) SEM (Scanning Electron Microscopy) picture of the quasi-2D experimental prototype composed of 3×3×3 unit-cells. e) SEM picture of the 3D experimental prototype consisting of 3×3×4 unit-cells.

Figure 2 :

 2 Figure 2: Deformation of the three examples of 2D systems considered in this work that correspond to different thicknesses of hinges. a) The first of the analyzed structures (case 1) where for the predeformed structure θ 0 = 90 • , θ 1 = 70 • , d 1 ≈ 9.3 µm, d 2 ≈ 6.1 µm. b) Case 2 with the initial parameters defined as θ 0 = 100 • , θ 1 = 70 • , d 1 ≈ 2.7 µm, d 2 ≈ 10.0 µm. c) Case 3 where for the predeformed system θ 0 = 100 • , θ 1 = 70 • , d 1 ≈ 2.6 µm, d 2 = 9.6 µm. d) Poisson's ratio measured for the compression along the z-axis calculated for the central topmost unit-cell. e) FEA results corresponding to the variation in the θ 0 and θ 1 angles throughout the deformation process. For all structures, l s ≈ 1200 µm. Mechanical metamaterials presented on panels a-b) and c) are composed of 3 × 3 × 3 and 3 × 3 × 2 unit-cells respectively. The remaining geometric parameters used for structures shown on panels a-c) were set to be the following: a) a 1 ≈ 77 µm, a 2 ≈ 78 µm, b ≈ 48 µm, d 3 ≈ 6.3 µm, d 4 ≈ 6.4 µm, b) a 1 ≈ 79 µm, a 2 ≈ 81 µm, b ≈ 48 µm, d 3 ≈ 12.7 µm, d 4 ≈ 10.4 µm and c) a 1 ≈ 77 µm, a 2 ≈ 79 µm, b ≈ 47 µm, d 3 ≈ 14.3 µm, d 4 ≈ 10.2 µm. For all mechanical metamaterials portrayed on panels a-c), the red and blue dashed lines correspond to the outline of the initial and deformed unit-cell respectively. All of the experimental pictures shown on panels a-c) were taken by means of the optical camera. Finally, the surface stress presented graphically for the FEA results on panels a-c) corresponds to the von Mises stress model implemented in the COMSOL Multiphysics software.

Figure 3 :

 3 Figure 3: The behavior of the type A and type B 3D hierarchical structures composed of 3 × 3 × 4 and 3 × 3 × 3 unitcells respectively. Both hierarchical models are subjected to the compression along the z-axis. a) Three different stages of the deformation process corresponding to the type A system. Provided pictures were captured by means of the optical camera and portrayed from the perspective of the xz plane. b) SEM picture of the type A prototype. c) The Poisson's ratio for compression along the z-axis calculated for the topmost row of unit-cells for both types of the considered 3D systems. The geometric parameters corresponding to the predeformed type A structure were the following: θ 0 = 90 • , θ 1 = 70 • , a 1 ≈ 33.2 µm, a 2 ≈ 33.8 µm, b ≈ 22.4 µm, d 1 ≈ 7.0 µm, d 2 ≈ 4.0 µm, d 3 ≈ 6.6 µm, d 4 ≈ 4.1 µm, l ≈ 88.7 µm and h ≈ 16.4 µm. On the other hand, for the type B system, these parameters were set to be as follows: θ 0 = 100 • , θ 1 = 70 • , a 1 ≈ 73.5 µm, a 2 ≈ 74.5 µm, b ≈ 44.4 µm, d 1 ≈ 2.6 µm, d 2 ≈ 9.2 µm, d 3 ≈ 14.5 µm, d 4 ≈ 12.5 µm, l ≈ 180 µm and h ≈ 33 µm . For pictures on panels (a-b), the blue dashed line indicates the dimensions of the undeformed structure in the xz plane.

Acknowledgements

This work was partly supported by the french RENATECH network and its FEMTO-ST technological facility. K.K.D. acknowledges the support of the Polish National Science Centre (NCN) in the form of the grant awarded as a part of the SONATINA 5 program, project No. 2021/40/C/ST5/00007 under the name "Programmable magneto-mechanical metamaterials guided by the magnetic field". K.K.D. acknowledges the financial support from the program of the Polish Ministry of Science and Higher Education under the name "Regional Initiative of Excellence" in 2019-2022, project no. 003/RID/2018/19, funding amount 11 936 596.10 PLN.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the corresponding author.


Avelino Álvarez-Ordóñez

Romain Briandet

Biofilms from a Food Microbiology Perspective: Structures, Functions, and Control Strategies

Keywords: biofilms, structure, control, food safety, food quality, bioprotection

Materials and equipment in food processing industries are colonized by surface-associated microbial communities called biofilms. In these biostructures microorganisms are embedded in a complex organic matrix composed essentially of polysaccharides, nucleic acids, and proteins. This organic shield contributes to the mechanical biofilm cohesion and triggers tolerance to environmental stresses such as dehydration or nutrient deprivation. Notably, cells within a biofilm are more tolerant to sanitation processes and the action of antimicrobial agents than their free living (or planktonic) counterparts. Such properties make conventional cleaning and disinfection protocols normally not effective in eradicating these biocontaminants. Biofilms are thus a continuous source of persistent microorganisms, including spoilage and pathogenic microorganisms, leading to repeated contamination of processed food with important economic and safety impact. Alternatively, in some particular settings, biofilm formation by resident or technological microorganisms can be desirable, due to possible enhancement of food fermentations or as a means of bioprotection against the settlement of pathogenic microorganisms.

In the last decades substantial research efforts have been devoted to unraveling mechanisms of biofilm formation, deciphering biofilm architecture, and understanding microbial interactions within those ecosystems. However, biofilms present a high level of complexity and many aspects remain yet to be fully understood. A lot of attention has been also paid to the development of novel strategies for preventing or controlling biofilm formation in industrial settings. Further research needs to be focused on the identification of new biocides effective against biofilm-associated microorganisms, the development of control strategies based on the inhibition of cell-to-cell communication, and the potential use of bacteriocins, bacteriocin-producing bacteria, phage, and natural antimicrobials as anti-biofilm agents, among others.

This research topic aims to provide an avenue for dissemination of recent advances within the "biofilms" field, from novel knowledge on mechanisms of biofilm formation and biofilm architecture to novel strategies for biofilm control in food industrial settings.

The research topic comprises three review articles, one perspective and 11 original research articles. Most of the contributions cover the most recent investigations on aspects related to the structures, architecture, and strategies for the control of biofilms formed by pathogenic or spoilage microorganisms on food processing surfaces, while two contributions are focused on the evaluation of biofilm formation by resident, technologically important or desirable microorganisms. In particular, they demonstrate that lactose induces formation of biofilm bundles, an increase in autoinducer-2 production in response to lactose, and an up-regulation of two gene operons responsible for extracellular matrix synthesis (e.g. eps and tapA).

In relation to Campylobacter jejuni biofilms, Brown and co-authors show in their contribution that extracellular DNA (eDNA) is an important component of C. jejuni biofilms formed on stainless steel surfaces (Brown et al.). The authors also evidence that eDNA may also contribute to the spread of antimicrobial resistance in C. jejuni. Finally, they report that degradation of eDNA by DNase I leads to rapid biofilm detachment, which shows promise for the control of C. jejuni biofilms in food industries. The research article by Turonova and co-authors reports that acclimation of two C. jejuni strains to oxygen-enriched conditions leads to a significant enhancement of biofilm formation during the early stages of the process, indicating that oxygen demand for biofilm formation is higher than for planktonic growth (Turonova et al.). The authors also identify the regulator CosR as a key protein in the maturation of C. jejuni biofilms. The research article by Bronnec and co-authors is aimed at evaluating the adhesion capacity and the ability to develop a biofilm of C. jejuni Bf, an atypical clinical isolate able to survive and grow under aerobic conditions (Bronnec et al.). The authors show that C. jejuni Bf can adhere to abiotic surfaces and human epithelial cells and can develop biofilms under both microaerobiosis and aerobiosis. They also conclude, from whole genome sequencing and transcriptomic analyses, that the behavior of this strain under aerobic atmosphere may result from the combination of different insertions and mutations and the modification of regulatory processes.

Two contributions are related to biofilms formed by strains of Staphylococcus spp. The perspective article by Oniciuc and co-authors shows that protein-based matrices are of relevance for the architecture of biofilms produced by Staphylococcus aureus strains isolated from food samples, as opposed to studies existing in the literature mentioning the predominance of exopolysaccharide-based matrices in biofilms formed by clinical and environmental isolates (Oniciuc et al.). Fagerlund and coauthors describe in their research article that the biofilm matrix composition has a significant impact on the efficacy of cleaning and disinfection agents against food associated Staphylococci (Fagerlund et al.). The authors show that some strains of Staphylococcus spp., able to form biofilms with a polysaccharide matrix, are resistant to benzalkonium chloride disinfectants, which are on the contrary effective for the removal of biofilms with a proteinaceous matrix.

Regarding biofilms formed by the foodborne pathogen Listeria monocytogenes, Zetzmann and co-authors report in their contribution that biofilms of L. monocytogenes are DNasesensitive at low ionic strength conditions, which might induce bacterial lysis and chromosomal DNA release (Zetzmann et al.). This suggests that DNase I treatment is an attractive option to prevent or remove L. monocytogenes biofilms in food processing environments, where low nutrient concentrations and increased osmotic pressures are frequently found conditions. Puga and co-authors evaluate by confocal laser scanning microscopy changes in spatial organization, biovolume, viable cell content and substratum surface coverage of biofilms produced on glass by L. monocytogenes in co-culture with Pseudomonas fluorescens (Puga et al.). The authors conclude: "when this dualspecies consortium develop biofilms on a solid surface, species interactions, cold stress and aging contribute to a more compact structure than the one built by P. fluorescens in single species biofilms."

Two review articles are related to strategies for the control of biofilms formed by pathogenic or spoilage microorganisms. Gutiérrez and co-authors examine environmental factors determining biofilm development in food processing equipment and discuss available information and future prospects on the use of bacteriophage-derived tools as successful disinfectants for the removal of biofilms (Gutiérrez et al.). On the other hand, Coughlan and co-authors discuss the problems associated with bacterial biofilms in the food industry and summarize recent strategies explored to inhibit biofilm formation, with special focus on those targeting quorum sensing (Coughlan et al.).

Two original research articles deal with biofilm formation by desirable microorganisms. The research article by Bastard and co-authors shows that Oenococcus oeni produces biofilms capable of efficient malolactic fermentation during winemaking and that O. oeni biofilms attached to oak can modulate wood-wine transfer of volatile aromatic compounds during wine fermentation and aging (Bastard et al.). Gómez and coauthors report in their contribution that probiotic strains can be good alternatives for the control of biofilm production by pathogenic bacteria in food-related environments (Gómez et al.). The authors evaluate the probiotic properties of several bacteriocinogenic and non-bacteriocinogenic lactic acid bacteria (LAB) and develop protective biofilms with some good probiotic candidates and test them for exclusion of L. monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium, obtaining promising results, with more than 6log reductions in viable counts being achieved with some of the LAB strains.

Finally, in the last contribution, Ostrov and co-authors develop a method (Cleaning-In-Place model system) to evaluate the effectiveness of cleaning agents in removal of biofilm derived spores from the surfaces of stainless steel in milking equipment in dairy farms (Ostrov et al.).

This editorial summarizes the articles published in this Research Topic, in the confidence that readers will find this information useful with the most recent research on microbial biofilms from a food microbiology perspective. We sincerely hope that this collection of papers will prompt further research and contribute to advance the knowledge on food-related biofilms and to develop novel or improved strategies of food safety and quality management.

  Various contributions deal with biofilms formed by strains of Bacillus spp. The review article by Majed and co-authors discusses the state-of-the-art on biofilms produced by Bacillus cereus, and by the two closely related pathogens, Bacillus thuringensis and Bacillus anthracis (Majed et al.). The review summarizes economic issues caused by B. cereus biofilms, the ecological and functional impact of biofilms in their lifecycle and management strategies implemented to control them. The research article by Hayrapeytan and co-authors shows the existence of intraspecies variability in the genome-encoded repertoire of iron-transporting systems and in the ability to grow and form biofilms in the presence of complex iron sources within B. cereus, which may influence B. cereus survival and persistence in food-related niches(Hayrapetyan et al.). Duanis-Assaf and co-authors report in their research article that may induce biofilm formation by Bacillus subtilis through a quorum sensing dependent (LuxS) pathway (Duanis-Assaf et al.).

Frontiers in Microbiology | www.frontiersin.org

November 2016 | Volume 7 | Article 1938

ACKNOWLEDGMENTS

We would like to thank the authors and reviewers for their valuable contributions and constructive criticisms to this special issue.

AUTHOR CONTRIBUTIONS

AÁ and RB designed and wrote the Editorial.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Álvarez-Ordóñez and Briandet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.