Amandine Gamble
email: amandine.gamble@gmail.com.

Romain Bazire

Karine Delord

Christophe Barbraud

Audrey Jaeger

Hubert Gantelet

Eric Thibault

Camille Lebarbenchon

Erwan Lagadec

Pablo Tortosa

Henri Weimerskirch

Jean-Baptiste Thiebot

Romain Garnier

Jérémy Tornos

Thierry Boulinier

Predator and scavenger movements among and within endangered seabird colonies: opportunities for pathogen spread

Keywords: conservation biology, disease ecology, dynamic space utilisation, individual heterogeneity, movement ecology, Pasteurella multocida, sentinel species, serology

1. The spatial structure of host communities is expected to constrain pathogen spread.

However, predators and/or scavengers may connect distant host (sub)populations when foraging. Determining whether some individuals or populations play a prominent role in the spread of pathogens is critical to inform management measures. effet le comportement alimentaire de telles espèces peut limiter l'efficacité des mesures locales malgré l'apparente structuration spatiale des populations affectée. Prédateurs et charognards peuvent ainsi représenter des cibles prioritaires pour les programmes de vaccination visant à limiter la dissémination d'agents infectieux pathogènes mais également être utilisés comme sentinelles pour en suivre la circulation et évaluer l'efficacité des programmes de contrôle.

2.

We explored movements and epidemiological status of brown skuas (Stercorarius antarcticus), the only avian terrestrial consumer native of Amsterdam Island (Indian Ocean), to assess whether and how they could be involved in the spread of the bacterium Pasteurella multocida, which recurrently causes avian cholera outbreaks in endangered albatross and penguin species breeding on the island.

3.

High proportions of seropositive and DNA-positive individuals for P. multocida indicated that skuas are highly exposed to the pathogen and may be able to transmit it. Movement tracking revealed that the foraging ranges of breeding skuas largely overlap among individuals and expand all along the coasts where albatrosses and penguins nest, but not on the inland plateau hosting the endemic Amsterdam albatross (Diomedea amsterdamensis).

4.

Considering the epidemiological and movement data, skua movements may provide opportunity for pathogen spread among and within seabird colonies.

Synthesis and applications.

This work highlights the importance of considering the behaviour and epidemiological status of predators and scavengers in disease dynamics because the foraging movements of individuals of such species can potentially limit the efficiency of local management measures in spatially-structured host communities. Such species could thus represent priority vaccination targets to implement efficient management measures aiming at limiting pathogen spread and also be used as sentinels to monitor pathogen circulation and evaluate the effectiveness of management measures.

Introduction

Infectious diseases threaten populations of many endangered wild species and are now recognized as a main threat relevant to conservation biology [START_REF] Young | Conservation, biodiversity and infectious disease: scientific evidence and policy implications[END_REF]. The structure of contact networks among hosts of infectious agents can strongly impact epidemiological dynamics [START_REF] Craft | Dynamics of a multihost pathogen in a carnivore community[END_REF][START_REF] Strona | The intrinsic vulnerability of networks to epidemics[END_REF], hence extinction probabilities [START_REF] Cleaveland | The role of pathogens in biological conservation[END_REF]. Some individuals or populations may occupy key positions in contact networks and contribute more than others to spreading infectious agents through their behaviour [START_REF] Lloyd-Smith | Superspreading and the effect of individual variation on disease emergence[END_REF][START_REF] Paull | From superspreaders to disease hotspots: linking transmission across hosts and space[END_REF][START_REF] Dougherty | Going through the motions: incorporating movement analyses into disease research[END_REF], notably in multi-host systems [START_REF] Craft | Dynamics of a multihost pathogen in a carnivore community[END_REF]. Such individuals or populations may thus constitute particularly relevant targets for disease control protocols aiming at interrupting transmission chains [START_REF] Rushmore | Network-based vaccination improves prospects for disease control in wild chimpanzees[END_REF][START_REF] Pepin | Contact heterogeneities in feral swine: implications for disease management and future research[END_REF][START_REF] Robinson | Model recommendations meet management reality: implementation and evaluation of a network-informed vaccination effort for endangered Hawaiian monk seals[END_REF].

Terrestrial predators and scavengers may effectively connect otherwise isolated colonies or social groups when foraging, potentially contributing to pathogen spread with conservation [START_REF] Craft | Dynamics of a multihost pathogen in a carnivore community[END_REF], public health [START_REF] Navarro | Pathogen transmission risk by opportunistic gulls moving across human landscapes[END_REF] or fundamental implications [START_REF] Boulinier | Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?[END_REF]. In wild communities subject to infectious disease outbreaks, deciphering the contact structure and examining the hosts' respective contributions in epidemiological dynamics is thus critical to develop control measures. The role of predator and/or scavenger species as potential spreaders of infectious agents is poorly documented because it requires targeted field efforts at the often unpredictable time of outbreaks [START_REF] Daversa | Infections on the move: how transient phases of host movement influence disease spread[END_REF]. For instance, the role of scavengers has been suspected in avian cholera, caused by the bacterium Pasteurella multocida (Pm), outbreaks [START_REF] Wille | A pelagic outbreak of avian cholera in North American gulls: scavenging as a primary mechanism for transmission[END_REF], but their actual contribution as spreaders still requires proper examination.

The recurrent outbreaks of avian cholera in seabirds on remote Amsterdam Island (Indian Ocean, 37°49'S, 77°33'E; [START_REF] Weimerskirch | Diseases threaten Southern Ocean albatrosses[END_REF][START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF] provide a unique opportunity to better understand the potential epidemiological role of a predator and scavenger species within a relatively simple host community (Figure 1). Avian cholera is a widespread disease severely threatening the viability of several avian populations [START_REF] Samuel | Effects of avian cholera on survival of Lesser Snow Geese Anser caerulescens: an experimental approach[END_REF][START_REF] Descamps | Avian cholera, a threat to the viability of an Arctic seabird colony?[END_REF]. On Amsterdam Island, Indian yellow-nosed albatrosses (Thalassarche carteri) have been recurrently hit by avian cholera outbreaks since the mideighties, potentially following the introduction of the pathogen through past human activities, such as animal farming, or accidental rodent introduction from visiting ships [START_REF] Micol | Restoration of Amsterdam Island, South Indian Ocean, following control of feral cattle[END_REF][START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. Infection by Pm causes septicaemia in nestlings, leading to their rapid death [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF][START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF] with important consequences on the breeding success of the local yellow-nosed albatross population. For instance, on Gough Island (which is rodent infested but considered avian cholera free), the breeding success of the Atlantic yellow-nosed albatross (Thalassarche chlororhynchos) is as high as 70% [START_REF] Cuthbert | Demography and population trends of the Atlantic yellow-nosed albatross[END_REF], while it has been below 10% most of the past twenty years on Amsterdam Island for the Indian yellow-nosed albatross [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. This example and others (e.g., [START_REF] Sebastiano | Food supplementation protects magnificent frigatebird chicks against a fatal viral disease[END_REF], illustrate that infectious diseases can represent an important, but often neglected, threat to seabird population viability. Avian cholera outbreaks on Amsterdam Island not only affect this globally-significant yellow-nosed albatross population [START_REF] Weimerskirch | Diseases threaten Southern Ocean albatrosses[END_REF], but are also suspected to cause mortality in two other endangered species: the sooty albatross (Phoebetria fusca) and the northern rockhopper penguin (Eudyptes moseleyi; [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF].

Despite the strong impact of avian cholera outbreaks on the endangered seabirds of Amsterdam Island [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF] and the availability of a vaccine proven to protect albatross nestlings [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF], no control measure has been implemented besides basic biosecurity measures. Indeed, relying on vaccination of nestlings to maintain the local yellow-nosed albatross population to its current size (~22000 pairs; [START_REF] Heerah | Important areas and conservation sites for a community of globally threatened marine predators of the Southern Indian Ocean[END_REF] would require to vaccinate thousands of nestlings every year, and may thus not represent an efficient conservation strategy. In contrast, interrupting transmission chains by treating individuals responsible for the among-colony circulation of the bacterium may help to protect some colonies with minimal efforts. Since Pm transmission occurs primarily through an oro-faecal route [START_REF] Samuel | Comparison of methods to detect Pasteurella multocida in carrier waterfowl[END_REF], it is unclear how Pm circulates locally. Indeed, seabirds on Amsterdam Island breed in spatially structured, mostly mono-specific, colonies (Figure S1.A.1) and albatrosses and penguins forage exclusively at sea [START_REF] Heerah | Important areas and conservation sites for a community of globally threatened marine predators of the Southern Indian Ocean[END_REF]. Hence, although contacts may be frequent within dense seabird colonies, contacts between individuals from different colonies may thus be rare besides potential limited prospecting movements [START_REF] Boulinier | Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?[END_REF].

Terrestrial predators and scavengers could however spread Pm across the island while foraging among seabird colonies undergoing outbreaks. Notably, the small population of brown skuas (Stercorarius antarcticus), the sole terrestrial vertebrate predators and scavengers native to the island, could disseminate the bacterium when foraging through shedding and/or by moving infected albatrosses and penguins [START_REF] Pietz | Feeding and nesting ecology of sympatric South Polar and brown skuas[END_REF]. Introduced brown rats (Rattus norvegicus) and house mice (Mus musculus; [START_REF] Micol | Restoration of Amsterdam Island, South Indian Ocean, following control of feral cattle[END_REF] could also carry and shed the bacterium, notably as they prey and scavenge on seabird chicks (Figure S1.A.2; [START_REF] Thiebot | Do introduced mammals chronically impact the breeding success of the world's rarest albatross[END_REF], although the distribution of resources and geographical barriers created by the island relief probably constrain the spatial scale of their contribution. In addition, the endemic Amsterdam albatross (Diomedea amsterdamensis) could also be at risk of exposure to Pm via foraging skuas because it nests a few kilometres inland from the yellow-nosed albatross colonies, although nestling die-offs have seldom been recorded in this species [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. Considering the wide range of feeding habits of skuas and the potential heterogeneity of foraging strategies among individuals [START_REF] Furness | The Skuas[END_REF], some individuals could play a prominent role in the circulation of infectious agents. In the skua population of Amsterdam Island, we examined the extent to which the individuals (1) are exposed to Pm, (2) forage on coastal versus inland areas, and (3) exploit exclusive feeding territories within the seabird colonies. We expected the skuas to forage mostly on the coasts where prey species breed in dense colonies (Figure 2a, scenarios 1 and 3), maximising feeding opportunities (Figure S1.A.2a), but also exposure risks to Pm. Further, we expected skuas to hold exclusive individual feeding territories (Figure 2a, scenarios 1 and 2; [START_REF] Pietz | Feeding and nesting ecology of sympatric South Polar and brown skuas[END_REF][START_REF] Trivelpiece | Feeding territories of brown skuas (Catharacta lonnbergi)[END_REF][START_REF] Votier | Reproductive consequences for Great Skuas specializing as seabird predators[END_REF], with restricted movements of some individuals potentially inducing heterogeneity in exposure to Pm and limited contacts among skuas (outside pairs, contacts are expected to occur mostly on foraging sites and clubs, i.e., sites where individuals gather outside breeding territories; [START_REF] Klomp | Variations in numbers of nonbreeding great skuas attending a colony[END_REF]. Testing these hypotheses should bring new insights on pathogen circulation in spatially structured host communities and help managers to design and implement efficient disease control protocols.

Materials and Methods

Study population

Around 60 pairs of brown skuas breed at low density yearly on Amsterdam Island's "Plateau des Tourbières" (PDT; Figure 1). Breeders generally lay two eggs in October/November and nestlings hatch in late November/early December and fledge 40-50 days later. Skuas attend clubs notably in the north ("Mare aux Éléphants" [MAE]) and the south-west (bottom of "Entrecasteaux" cliffs). Amsterdam island holds important populations of Indian yellow-nosed albatrosses, sooty albatrosses (~400 pairs; [START_REF] Heerah | Important areas and conservation sites for a community of globally threatened marine predators of the Southern Indian Ocean[END_REF] and northern rockhopper penguins (~12000 pairs), all breeding in mostly mono-specific colonies on the south-west coastal cliffs between August and May (Figure 1). The 40-50 pairs of Amsterdam albatrosses nest biennially further inland, on the northern part of the PDT (Figure 1). A few small Procellariiformes are also present on the island in very low numbers [START_REF] Micol | Restoration of Amsterdam Island, South Indian Ocean, following control of feral cattle[END_REF]. All around the island, subantarctic fur seals (Arctocephalus tropicalis) calve on the beaches in December [START_REF] Guinet | Long term population changes of fur seals Arctocephalus gazella and Arctocephalus tropicalis on subantarctic (Crozet) and subtropical (St. Paul and Amsterdam) islands and their possible relationship to El Niño Southern Oscillation[END_REF], also providing food opportunities for skuas (placentas and dead pups). Introduced brown rats and house mice are often observed in seabird colonies (Figure S1.A.2). Feral cats (Felis catus) were also introduced on the island [START_REF] Micol | Restoration of Amsterdam Island, South Indian Ocean, following control of feral cattle[END_REF], but are rarely observed in seabird colonies.

Field sampling

During three breeding seasons (2011-2012, 2015-2016, 2016-2017), blood samples (1 mL from the metatarsal vein using heparinized syringes) and cloacal swabs (using sterile cotton tips) were collected from 66 adults and 9 nestlings of brown skuas captured on their nests in the southern part of PDT or in clubs at Entrecasteaux and MAE between November and January (Table 1). Breeders handled in 2015-2016 and breeders and club attendants handled in 2016-2017 were marked with leg rings for individual identification. Red blood cells and plasma were separated by centrifugation a few hours after collection. Swabs were stored in 0.5 mL of a lysis buffer (RNA NOW ® , BIOGENTEX, USA, in 2011-2012; Longmire buffer the following years; [START_REF] Longmire | Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family Falconidae[END_REF]. Samples were kept at -20°C in the field, then stored at -20°C (plasma) or -80°C (swabs) until analysis.

Immunological assays

Pm-specific antibody levels in plasma samples of skuas were measured using two immunoassays in order to ascertain past exposure to Pm: an enzyme-linked immunosorbent assay (ELISA; ID Screen ® Pasteurella multocida Chicken and Turkey Indirect, IDvet, France; with the positivity threshold determined following [START_REF] Garnier | Interpreting ELISA analyses from wild animal samples: some recurrent issues and solutions[END_REF] and a microagglutination test (MAT; SEROPAST ® , Ceva-Biovac, France). Technical details are given in Appendix S1.B.

Molecular detection of Pm

Total nucleic acids were extracted following the RNA NOW ® isolation and purification protocol for skua cloacal swabs preserved in RNA NOW ® , and with the QIAamp cador Pathogen Mini ® kit (QIAGEN, USA) for skua cloacal swabs preserved in Longmire lysis buffer.

Pm DNA was detected with a real-time polymerase chain reaction (RT-PCR) targeting the strain previously detected in a dead sooty albatross on Amsterdam Island [START_REF] Jaeger | Exploring the infection dynamics of a bacterial pathogen on a remote oceanic island reveals annual epizootics impacting an albatross population[END_REF].

Technical details are given in Appendix S1.B.

Movement tracking

In 2015-2016 and 2016-2017, 18 breeding skuas captured on the southern PDT were also equipped with solar-powered GPS-UHF devices with a remote data download link (GPS-UHF Harrier-L ® , Ecotone, Poland). The GPS acquisition frequency was set at 2 to 5 min and altitude above sea level was also recorded in 2015-2016 (see Appendix S1.C for more details).

Loggers were deployed during the early chick-rearing period (late November/early December), when skuas' energetic needs are at their highest [START_REF] Furness | The Skuas[END_REF] and avian cholera outbreaks occur in albatross colonies [START_REF] Weimerskirch | Diseases threaten Southern Ocean albatrosses[END_REF][START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF]. Sufficient data for spatial analysis (> 24h) were collected from 13 individuals, with data spanning 12-102 days Using the GPS data, the intensity (spatial distribution of the mean residence time per visit of each area, i.e., areas where an individual tends to spend more time) and recursion (spatial distribution of the number of visits in each area, i.e., areas that an individual tends to repeatedly visit) distributions were computed to get a dynamic picture of space utilisation [START_REF] Benhamou | Beyond the utilization distribution: Identifying home range areas that are intensively exploited or repeatedly visited[END_REF]. The overlap of the patches recursively used by different individuals was visually examined to inform on the existence of exclusive feeding territories in this population. To estimate the intensity and recursion distributions, non-overlapping virtual circles of 50m in radius (referred to as "areas") were delimited. A new visit to an area occurred each time the tracked individual re-entered it after a time lag spent outside the area longer than 10 min and remained in that area for at least 15 min (to exclude cases in which birds went through an area without using it) using the BRB|MKDE program (for Biased Random Bridges for Movement-based Kernel Density Estimation; [START_REF] Benhamou | Dynamic approach to space and habitat use based on biased random bridges[END_REF]. All data were analysed and represented after exclusion of locations recorded within the nesting area of the tracked individuals unless indicated otherwise. Resulting distributions were visualized in QGIS 2.18.21.

Monitoring of yellow-nosed albatrosses

In order to assess whether skuas were present in coastal seabird colonies during avian cholera epizootics, the proportions of yellow-nosed albatross nestlings surviving and excreting Pm DNA in a subcolony of Entrecasteaux were used as a proxy for the progress of the epizootics. Pm DNA excretion was assessed using the same design used in brown skuas.

Details and sample sizes are given in Appendix S1.D.

Statistical analyses

All statistical analyses were run using R 3.6.1 and the script available in Appendix S3.

Seroprevalences were calculated as the proportion of seropositive individuals among tested individuals each year. Differences in antibody levels among locations within a year or among years in a location were investigated using Wilcoxon tests with a Bonferroni correction and a 5% significance level.

Results

Detection of Pm and anti-Pm antibodies

Specific antibodies were detected in all but two plasma samples from adult brown skuas by both MAT and ELISA, resulting in seroprevalence varying between 80% and 100% depending on the site and year (Table 1). In 2016-2017, based on the ELISA results, antibody levels were significantly lower in MAE than in Entrecasteaux and PDT (both p<0.01; Table S1.B.1). All other pairwise comparisons were not statistically different (Figure 3). The same results were obtained from the MAT data. Pm DNA was detected in cloacal swabs of adults from PDT and Entrecasteaux, but not from MAE (Table 1). The proportion of Pm positive samples was generally low (≤33%), except for PDT in 2016-2017 with 8/10 positive individuals.

Regarding nestlings, neither specific antibodies nor Pm DNA were detected in samples from 2011-2012. In 2016-2017, Pm DNA was detected in three seronegative nestlings; one other nestling was seropositive but not excreting Pm DNA (Table 1; Figure S1.E.1).

Individual movement tracking

Both years, breeding skuas moved along the western coast (south to north-east) of the island, where fur seals, albatrosses and penguins breed (Figure 4). Some individuals visited the whole western coast within 24 hours (Figure S1 S1.C.2), notably during avian cholera outbreaks (Figure S1.D.1), and represented the only recursively visited site for four of them. Other sites recursively visited included the northern part of the south-west cliff, harbouring large seabird and fur seal colonies, the north coast, hosting another large fur seal colony, and the south plain where seabirds and fur seals are rare, but rodent populations may be present. Note that all the tracked individuals were seropositive and Pm DNA was detected in 8/13 birds (Table S1.C.2) at the time of logger deployment.

Discussion

Here, we explored different scenarios of foraging strategies of a predator and scavenger, the brown skua, in relation to the circulation of a pathogen within a spatiallystructured community of endangered seabirds. Movement data suggest that skuas may interconnect several seabird colonies during avian cholera epizootics, potentially contributing to pathogen spread. Our study allowed to characterise biological processes relevant to pathogen circulation, such as the fact that skuas do not hold foraging territories, a necessary first step to build mechanistic models of eco-epidemiological dynamics and explore potential control measures [START_REF] Daversa | Infections on the move: how transient phases of host movement influence disease spread[END_REF][START_REF] Dougherty | Going through the motions: incorporating movement analyses into disease research[END_REF].

Skua exposure and infection by Pm and implications for epidemiological surveillance

We report the first detection of anti-Pm antibodies in skuas and show that the population of brown skuas from Amsterdam Island is highly exposed to this bacterium. The proximity of Entrecasteaux cliffs, where skuas may be exposed to Pm through feeding on infected albatrosses and penguins (Figure S1.A.2a), may intensify exposure and explain the high specific antibody levels measured in individuals sampled at the Entrecasteaux club or breeding on PDT [START_REF] Pepin | Inferring infection hazard in wildlife populations by linking data across individual and population scales[END_REF]. Because detailed antibody kinetics are not available, it is not possible to estimate the timing of infection from serological data. Nevertheless, the detection of PCR-positive birds at Entrecasteaux club or breeding on PDT reveals ongoing infection at the time of sampling. In contrast, skuas attending the MAE club may forage preferentially on nearby fur seal colonies, potentially minimising their exposure to Pm, which would explain their lower antibody levels and the absence of PCR-positive birds at this site.

More generally, the important proportion of seropositive but PCR-negative adult skuas suggests that specific antibodies may persist past the infection period, as commonly observed in acute infections [START_REF] Pepin | Inferring infection hazard in wildlife populations by linking data across individual and population scales[END_REF]. [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF] reported the circulation of a unique Pm strain among sooty and yellow-nosed albatrosses, but technical constraints for field microbiology (e.g. low availability of fresh carcasses, difficulty to obtain bacterial isolates from non-invasive approaches) limited the sample sizes. While it is not possible to confirm that the epizootic Pm strain killing albatrosses is circulating in skuas, our current understanding of the system supports the circulation of a common Pm strain circulating in the different species of the island. Future works should aim at sequencing more Pm isolates from different species of the island to confirm this hypothesis.

Interestingly, detection of specific antibodies in the majority of PCR-negative adults also suggests that a large proportion may survive Pm exposure, contrasting with the high mortality rate reported during previous outbreaks in Antarctica [START_REF] Parmelee | Fowl cholera outbreak among brown skuas at Palmer Station[END_REF][START_REF] Leotta | Outbreaks of avian cholera in Hope Bay, Antarctica[END_REF]. This heterogeneity is not surprising given the known variability of Pm virulence in different hosts [START_REF] Christensen | Fowl cholera[END_REF]. Moreover, the low mortality rate of adult skuas on Amsterdam Island is similar to that of adult yellow-nosed albatrosses on that island [START_REF] Rolland | Assessing the impact of fisheries, climate and disease on the dynamics of the Indian yellow-nosed albatross[END_REF]Gamble, Ganier et al., 2019), but the higher seroprevalence in skuas suggests different rates of exposure and/or persistence of antibody levels. If skuas are highly exposed but resistant or tolerant to infection by Pm, they could also be involved in the maintenance of the pathogen on the island. This result also shows that skuas are potentially good sentinels to detect the circulation of Pm, especially using serology [START_REF] Halliday | A framework for evaluating animals as sentinels for infectious disease surveillance[END_REF]. Hence, implementing long-term serosurveys targeting such species can be especially useful for the acquisition of baseline data on (pathogenic and non-pathogenic) infectious agent circulation and for the early detection of pathogen circulation. Indeed, in longlived species such as seabirds, monitoring protocols based only on breeding pair counts can take several years before capturing juvenile mortality events because of late recruitment.

Movements and disease transmission in structured communities

Apex predator movements have been well studied in relation to foraging during the breeding season, but their implications for infectious agent circulation have only recently been considered [START_REF] Boulinier | Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?[END_REF][START_REF] De Souza Petersen | First detection of avian influenza virus (H4N7) in Giant Petrel monitored by geolocators in the Antarctic region[END_REF]. As observed elsewhere [START_REF] Carneiro | Foraging behaviour and habitat use by brown skuas Stercorarius lonnbergi breeding at South Georgia[END_REF][START_REF] Pietz | Feeding and nesting ecology of sympatric South Polar and brown skuas[END_REF], skuas breeding on Amsterdam Island foraged almost exclusively onshore, mostly along the coastal cliffs harbouring dense seabird colonies with high feeding opportunities. This population may thus regularly be in contact with yellow-nosed and sooty albatrosses, rockhopper penguins and subantarctic fur seals during avian cholera outbreaks. Interestingly, several individuals recursively used different patches along the western coasts, corresponding to different seabird and fur seal colonies: this may reflect particular opportunities for the dissemination of infectious agents. In addition, as opposed to other populations [START_REF] Trivelpiece | Feeding territories of brown skuas (Catharacta lonnbergi)[END_REF][START_REF] Pietz | Feeding and nesting ecology of sympatric South Polar and brown skuas[END_REF][START_REF] Votier | Reproductive consequences for Great Skuas specializing as seabird predators[END_REF]; but see [START_REF] Carneiro | Foraging behaviour and habitat use by brown skuas Stercorarius lonnbergi breeding at South Georgia[END_REF], breeding skuas on Amsterdam Island did not seem to hold individual-exclusive feeding territories, as supported by the large space utilisation overlap among individuals. The high food availability in large breeding colonies of vertebrates may explain the absence of such territories on Amsterdam Island. Accordingly, the absence of individual-exclusive feeding territories associated with high seroprevalences suggests that, if breeding individuals contribute to Pm circulation on the island, their role could be rather homogeneous (Figure 2), contrary to other systems [START_REF] Marchand | Sociospatial structure explains marked variation in brucellosis seroprevalence in an Alpine ibex population[END_REF][START_REF] Dougherty | Going through the motions: incorporating movement analyses into disease research[END_REF]. However, other possible sources of heterogeneity among individuals remains to be explored, such as variations in the duration or intensity of shedding.

We did not observe breeding skuas foraging in the small and low-density breeding population of the Amsterdam albatross, suggesting that they may not connect this population to others (Figure 2b). This is consistent with the high breeding success of this albatross population over the last decades [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. Although a few skua pairs nest among Amsterdam albatrosses (Figure 1), contacts between the two species may be rare if skuas forage only on the coast, as movements around the nests are usually limited. However, considering the flexibility of skuas' foraging behaviour and potential behavioural differences between breeders and non-breeders, some individuals may occasionally visit this colony, especially if environmental conditions and food availability change [START_REF] Carneiro | Flexibility in foraging strategies of Brown Skuas in response to local and seasonal dietary constraints[END_REF]. Such changes, modifying the host space-utilisation, can have important consequences on the dynamics of infectious agents [START_REF] Merkle | Linking spring phenology with mechanistic models of host movement to predict disease transmission risk[END_REF][START_REF] Giles | Environmental drivers of spatiotemporal foraging intensity in fruit bats and implications for Hendra virus ecology[END_REF]. Long-term monitoring of these dynamics is essential to better understand pathogen circulation, and design robust management options.

Maintenance and circulation of infectious agent

The population of brown skuas may play a key role in the circulation of Pm on Amsterdam Island, but the complete maintenance community (i.e., the set of connected host populations that together can maintain the pathogen over the long term, notably in winter when most seabirds are absent of the island; [START_REF] Viana | Assembling evidence for identifying reservoirs of infection[END_REF] remains to be functionally characterized. Rodents may be good candidates as local maintenance hosts of Pm, but little data are yet available to examine this hypothesis. Rodents can reach high densities and are present year-round; they may feed on dead seabird nestlings and are preyed upon by skuas (Figure S1.A.2b,d). In addition, as observed in poultry [START_REF] Curtis | Transmission of Pasteurella multocida infection from the brown rat (Rattus norvegicus) to domestic poultry[END_REF], rodents may directly transmit Pm through movements within colonies and biting of live nestlings [START_REF] Thiebot | Do introduced mammals chronically impact the breeding success of the world's rarest albatross[END_REF]Figure S1.A.2c). Hence, spatio-temporal aspects of rodent exposure to Pm and subsequent shedding need to be explored from an eco-epidemiological standpoint [START_REF] Lloyd-Smith | Should we expect population thresholds for wildlife disease?[END_REF][START_REF] Guzzetta | Population dynamics of wild rodents induce stochastic fadeouts of a zoonotic pathogen[END_REF]. Because the home range radius of rodents is likely much smaller than that of skuas, rodents could be important in the within-colony spreading of Pm, while skuas may play a key role at a larger spatial scale. These elements suggest potentially complex epidemiological networks involving several bridge hosts [START_REF] Caron | Bridge hosts, a missing link for disease ecology in multi-host systems[END_REF] linking cliff-nesting seabirds to Amsterdam albatrosses. Skuas could move Pm from the costal cliffs to PDT, where a few breed in proximity to Amsterdam albatrosses.

Foraging rodents could then move the bacterium from those skuas to the Amsterdam albatrosses. More generally, this stresses the importance of considering processes occurring at nested spatial scales, and epidemiological networks in their entirety when exploring chains of transmission [START_REF] Boulinier | Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?[END_REF][START_REF] Daversa | Infections on the move: how transient phases of host movement influence disease spread[END_REF]. In the case of Amsterdam Island, future work should examine the role of introduced rodents in the maintenance and small-scale circulation of avian pathogens.

Implications for conservation

Our study revealed that the movements of potential bridge hosts (here, skuas and potentially rodents) may reduce the efficiency of local actions aiming at controlling multi-host infectious agents. For instance, locally intensive vaccination programs targeting albatrosses (e.g., [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF][START_REF] Gamble | Dynamics of antibody levels against avian cholera after natural exposure and vaccination of albatrosses: disease ecology implications[END_REF] combined to rodent population control would seem feasible in accessible seabird colonies. However, such attempts would likely fail to control the pathogen because, although the system appears highly spatially structured, skuas could re-introduce the pathogen from inaccessible, hence non-treated, seabird colonies. In such a system, efficient disease control measures would likely need to interrupt the transmission network by targeting bridge hosts by, for instance, vaccinating native terrestrial predators (here skuas) with a vaccine blocking transmission and eradicating introduced rodents. However, we do not recommend culling native terrestrial predators as there are growing evidences that scavenging contribute in disease controls (e.g., [START_REF] Sage | Do scavengers prevent or promote disease transmission? The effect of invertebrate scavenging on Ranavirus transmission[END_REF] and the culling of a native species would raise strong ethical issues. In the case of avian cholera on Amsterdam island, an autogenous vaccine has proven efficient to protect yellownosed albatross nestlings [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF], but whether it blocks transmission remains to be investigated. The direct negative impact of introduced rodents on seabird populations is well documented and their eradication is seen as a priority for seabird conservation (see [START_REF] Duron | Control of invasive rats on islands and priorities for future action: rats on islands[END_REF] for a review). In contrast, their role in epidemiological dynamics has rarely been explored, but its potential is highlighted by our results. Implementing the eradication of introduced rodent populations on Amsterdam Island in parallel to the epidemiological monitoring of seabird populations, would provide a unique opportunity to semi-experimentally assess their role in the eco-epidemiological dynamics while representing a significant management action in itself.

When deciding what management measure to implement in a situation such as the one on Amsterdam Island, modelling approaches can enable the stakeholder to clearly outline the management objectives, the available means to reach them, and the level of uncertainty in the parameters underlying the dynamics of the system. Indeed, comparing the potential benefits of different management strategies is not as straightforward as it may seem and we encourage stakeholders to work hand in hand with disease ecologists and modellers, even in an apparently simple situation such as that on Amsterdam Island. For instance, compartmented epidemiological models represent attractive options to assess the necessary level of rodent population control [START_REF] Mariën | Evaluation of rodent control to fight Lassa fever based on field data and mathematical modelling[END_REF] or vaccination coverage and protocols [START_REF] Haydon | Low-coverage vaccination strategies for the conservation of endangered species[END_REF][START_REF] Baker | Optimizing spatial and seasonal deployment of vaccination campaigns to eliminate wildlife rabies[END_REF] to efficiently benefit endangered species while accounting for their particular life-history traits [START_REF] Garnier | Maternal antibody persistence: a neglected life-history trait with implications from albatross conservation to comparative immunology[END_REF]. Network models based on (direct or indirect) contact networks at the inter-specific [START_REF] Craft | Dynamics of a multihost pathogen in a carnivore community[END_REF][START_REF] Woodroffe | Risk of contact between endangered African wild dogs Lycaon pictus and domestic dogs: opportunities for pathogen transmission[END_REF] and intra-specific levels [START_REF] Rushmore | Network-based vaccination improves prospects for disease control in wild chimpanzees[END_REF][START_REF] Pepin | Contact heterogeneities in feral swine: implications for disease management and future research[END_REF][START_REF] Robinson | Model recommendations meet management reality: implementation and evaluation of a network-informed vaccination effort for endangered Hawaiian monk seals[END_REF] could be used to explore the benefits of targeting potential super-spreading individuals or group of individuals for vaccination or population control. Models also allow to explore sources of heterogeneity in pathogen maintenance and transmission [START_REF] Marchand | Sociospatial structure explains marked variation in brucellosis seroprevalence in an Alpine ibex population[END_REF][START_REF] Dougherty | Going through the motions: incorporating movement analyses into disease research[END_REF][START_REF] Robinson | Model recommendations meet management reality: implementation and evaluation of a network-informed vaccination effort for endangered Hawaiian monk seals[END_REF]. Finally, management should ideally be conducted in an adaptive dynamic framework [START_REF] Keith | Uncertainty and adaptive management for biodiversity conservation[END_REF], requiring a design allowing to track eco-epidemiological variables, notably through the monitoring of sentinel species [START_REF] Halliday | A framework for evaluating animals as sentinels for infectious disease surveillance[END_REF], before and after interventions [START_REF] Viana | Assembling evidence for identifying reservoirs of infection[END_REF]. The present paper provide key elements to fuel such modelling approaches.

Authors' contributions. TB conceived the idea of this work; TB, JT, HW, KD and CB designed the study; JT, AG and J-BT collected the data; HG and ET developed the MAT; AG, AJ, CL, PT and EL performed the serology and molecular analyses; AG analysed the epidemiological data; AG, RB and J-BT analysed the tracking data; AG and TB led the writing of the manuscript. All authors contributed substantially to the drafts and gave final approval for publication.

Résumé 1 .

 1 La circulation d'agents infectieux dans les populations sauvages peut être fortement contrainte par la structuration spatiale de leurs communautés d'hôtes. Cependant, de par leurs comportements alimentaires, certains prédateurs et/ou charognards peuvent connecter des (sous-)populations spatialement éloignées. Identifier les individus ou populations susceptibles de jouer un rôle prépondérant dans la dissémination d'agents infectieux pathogènes est un élément clé pour la compréhension, et éventuellement le contrôle, des dynamiques épidémiologiques dans les populations sauvages. 2. Dans ce contexte, nous avons suivi les déplacements et statuts épidémiologiques de labbes subantarctiques (Stercorarius antarcticus), uniques prédateurs et charognards terrestres aviaires natifs de l'île Amsterdam (Océan Indien), afin d'évaluer leur implication dans la dissémination de Pasteurella multocida, la bactérie responsable d'épizooties récurrentes de choléra aviaire affectant les albatros et gorfous de l'île. 3. Les fortes proportions d'individus positifs à P. multocida par sérologie et PCR indiquent que les labbes sont très exposés à la bactérie et susceptibles de la transmettre. Par ailleurs le suivi des déplacements d'individus reproducteurs a révélé que les zones de recherche alimentaire individuelles des labbes se superposent et s'étendent tout le long de la partie de côte occupée par les albatros et les gorfous. Au contraire, le plateau central où se reproduit l'albatros d'Amsterdan (Diomedea amsterdamensis), espèce endémique de l'île, ne semble pas visité par les labbes en reproduction. 4. Compte tenu des données épidémiologiques et écologiques présentées les labbes sont susceptibles de contribuer à dissémination d'agents infectieux au sein et entre des colonies d'oiseaux marins menacés d'extinction. 5. Synthèse et applications. Cette étude met en évidence l'importance de prendre en considération le comportement et le statut épidémiologique des prédateurs et charognards lors de la mise en place de programmes de contrôle de dynamiques épidémiologiques. En

(

  median [25 and 75% quartiles] = 54 [47;85]; TableS1.C.1), covering most of the chick-rearing period for the majority of the tracked individuals. A bootstrap analysis estimated this sample to adequately represent space use of the skua population breeding on southern PDT during this period[START_REF] Lascelles | Applying global criteria to tracking data to define important areas for marine conservation[END_REF]; see Appendix S1.C and FigureS1.C .1).

  .C.3, Appendix S2). Breeding skuas visited the surroundings of Entrecasteaux and MAE clubs. When travelling along the cliffs, skuas exploited a large altitudinal gradient, suggesting that they potentially visited fur seals, penguins and albatross colonies (Figures S1.C.2, S1.C.3). However, none of the tracked individuals visited the Amsterdam albatross breeding area. Recursively exploited areas largely overlapped among the tracked individuals (Figures 5, S1.C.4). Entrecasteaux cliffs were intensively and recursively exploited by all the tracked individuals (Figures 5, S1.C.4, S1.C.5, Table

Figure 1 .

 1 Figure 1. Breeding sites of colonial vertebrates on Amsterdam Island (37°49'S, 77°33'E).

Figure 3 .

 3 Figure 3. Evidence of high exposure of brown skuas to Pasteurella multocida: anti-Pm

Figure 4 .

 4 Figure 4. Space utilisation by breeding brown skuas. Left: distribution of space utilisation

Figure 5 .

 5 Figure 5. Recursive space utilisation by breeding brown skuas equipped with GPS-UHF

Acknowledgements. We thank Jacob González-Solís for providing the supplementary samples from the Falkland/Malvinas Islands, Baudouin Des Monstiers, Augustin Clessin, Jérémy Dechartre, Raul Ramos, Simon Benhamou and Olivier Duriez for discussions and Nicolas Giraud, Marine Bely and Vincent Bourret for help in the field. We also thank Yann Rantier (IPEV SUBANTECO-136/ECOBIO, Rennes) for the 3D map production, Stéphanie Lesceu and Khadija Mouacha (IDvet, France) for their help in adapting the ELISA protocol and anonymous reviewers for useful comments. This work was funded by the French Polar Institute (IPEV ECOPATH-1151 and ORNITHOECO-109), ANR EVEMATA (11-BSV7-003), Réserve Naturelle Nationale des Terres Australes Françaises and Zone Atelier Antarctique. This paper is a contribution of the Plan National d'Action Albatros d'Amsterdam. AG received a PhD fellowship from the French Ministry of Research and was later supported by NSF (DEB-1557022) and SERDP (RC-2635), and CL acknowledges support from INSERM -Université de La Réunion. The experimental design was approved by the Comité de l'Environnement Polaire (TAAF A-2015-107, A-2016-80).

Data availability statement. Data are available on the OSU-OREME Repository https://doi.oreme.org/b4b4a2e1-3d00-45b7-b3b2-2ca05d054346 [START_REF] Gamble | ECOPATH: brown skua Pasteurella multocida epidemiological data and GPS tracking data[END_REF]


Nicolas Martin
email: nicolas.martin@crpp.cnrs.fr

Liangfei Tian

Dan Spencer

Angélique Coutable-Pennarun

J L Ross Anderson

Stephen Mann
email: s.mann@bristol.ac.uk

J L Ross

Photoswitchable phase separation and oligonucleotide trafficking in DNA coacervate microdroplets

come   L'archive ouverte pluridisciplinaire

new approaches for the photo-induced control of DNA coacervation and oligonucleotide compartmentalization in space and time, and provide a step towards the dynamic regulation of membrane-free synthetic protocells.

Light-responsive coacervate micro-droplets were produced in water in the dark as a turbid suspension via spontaneous liquid-liquid phase separation associated with the stoichiometric charge neutralization of short dsDNA strands (5 mM nucleobase concentration, <200 base pairs (bp), Figure S1) in the presence of trans-azoTAB (5 mM) and sodium chloride (100 mM). Optical microscopy images of the suspension revealed the presence of discrete polydisperse spherical droplets approximately 1 to 10 μm in diameter (Figure 1b) that coalesced on contact, consistent with a liquid-like state (Figure S2 and SI Note 1), and that were able to selectively sequester various solutes (Figure S3). The driving forces responsible for droplet assembly included a combination of electrostatic and hydrophobic interactions, together with partial intercalation of the molecular photoswitch between the dsDNA base pairs [39][40][41] (Figure S4). Although the trans-azoTAB/dsDNA micro-droplets were stable across a wide range of ionic strengths, pHs and temperatures (Figures S4 andS5), they readily disassembled within a few seconds when exposed to UV light (λ = 365 nm, Figure 1c, Movie S1 and Figure S6). We attributed the light-activated droplet disassembly to the reversible photo-isomerisation of trans-azoTAB into the non-planar cis-azoTAB metastable isomer, which exhibits a lower affinity for dsDNA [39][40][41] (Figure S4 andS7). Coacervate droplets could be re-assembled by irradiating solutions of cis-azoTAB/dsDNA with blue light (λ = 450 nm), which re-generated the thermodynamically stable trans-azoTAB isomer (Figure 1d, Movie S2 and Figure S6). Significantly, the kinetics of the photo-switchable phase separation could be finely modulated by adjusting the light intensity, and the process repeated over numerous cycles without any apparent degradation (Figure S8).

The dynamics of droplet disassembly and re-assembly were exploited for the correlated release and recapture of sequestered guest molecules. For example, doping dark-adapted trans-azoTAB/dsDNA droplets with a fluorescent single-stranded oligonucleotide (TAMRA-ssDNA, 23 nucleobases) and exposing the loaded droplets to UV light showed a gradual and quantitative release of the oligonucleotide into the surrounding aqueous phase (Figures 1e,f). Subsequent exposure of the cis-azoTAB/dsDNA solution to blue light reassembled the droplets and recaptured approximately 80% of the oligonucleotide (Figures 1e,f). Alternatively, temporal of DNA phase separation was achieved by controlling the spontaneous thermal relaxation of cis-to trans-azoTAB in the dark. Optical microscopy images of a UV-equilibrated cis-azoTAB/dsDNA solution incubated in the dark confirmed a gradual increase in the number and size of coacervate droplets (Figure 1g). The corresponding changes in turbidity showed a sigmoidal increase, consistent with a kinetically slow nucleation step followed by rapid growth up to the saturation limit (Figure 1h). Significantly, the onset time for phase separation was strongly correlated with the temperature following a simple Arrhenius law (Figure S9 and Table S2), demonstrating that DNA phase separation could be robustly programmed in time. For example, by exposing trans-azoTAB/dsDNA droplets to cycles comprising short periods of UV light irradiation followed by storage in the dark at a specific temperature, predictable oscillations in the time-dependent concentration of free TAMRA-ssDNA could be realized (Figure S10).

Given the above observations, we sought to increase the networking capacity of the photoactive coacervate system by undertaking experiments geared towards controlling the photo-induced trafficking of captured oligonucleotides. We devised an experimental system in which oligonucleotide-containing photo-responsive coacervate droplets and payload-free non-photo-responsive droplets were physically separated by a semipermeable barrier and then used UV light to induce transfer of the oligonucleotide between the two populations (Figure 2a). Specifically, trans-azoTAB/dsDNA droplets containing TAMRA-ssDNA and non-photo-responsive poly(diallyldimethylammonium chloride) (PDDA)/dsDNA droplets were loaded in the dark into two separate sample chambers arranged on a capillary slide and separated by an agarose hydrogel barrier. Corresponding optical and fluorescence microscopy images showed the presence of discrete droplets in both populations but only the trans-azoTAB/dsDNA droplets displayed red fluorescence due to retention of TAMRA-ssDNA (Figures 2b,c). In contrast, when the capillary slide was irradiated with UV light, dissolution of the lightresponsive droplets resulted in the release of the oligonucleotide, slow diffusive transfer across the hydrogel barrier and graded capture of the payload in the population of PDDA/dsDNA droplets (Figure 2d and Figure S11). We employed a related strategy to control the light-activated transfer and manipulation of genetic information within dispersed binary populations of spatially interacting photo-responsive coacervate microdroplets. Populations of trans-azoTAB/dsDNA coacervate droplets containing fluorescence resonance energy transfer (FRET)-paired complementary single strands of TAMRA-ssDNA or carboxyfluorescein-labelled ssDNA (FAM-ssDNA, 23 nucleobases) were mixed in the same observation chamber and sequentially exposed to UV and blue light (Figure 3a and Table S3). Confocal fluorescence microscopy revealed the coexistence of two distinct droplet populations that exhibited either red fluorescent (TAMRA-ssDNA payload) or green fluorescent (FAM-ssDNA payload) (Figure 3b). No spontaneous ssDNA exchange between the droplets was observed for samples kept in the dark. In comparison, disassembly of the droplets under UV light followed by reassembly after exposure to blue light produced droplets that exhibited both green and red fluorescence (Figure 3c), indicating that the complementary strands became co-localized via reversible photo-induced microphase separation. Moreover, the co-localized strands displayed increased FRET fluorescence, which was attributed to hybridization of the released oligonucleotides either prior to droplet re-assembly or within the droplets after colocalization, or both (Figure 3d). Given the ability to transfer genetic polymers between different populations of coacervate droplets, we used a focused UV beam to induce the localized trafficking of DNA between single neighbouring droplets (Figure 3e). For this purpose, a mixture of unloaded and TAMRA-ssDNA-loaded trans-azoTAB/dsDNA coacervate droplets was prepared and imaged by confocal fluorescent microscopy combined with bright-field microscopy, which confirmed the co-existence of two types of coacervate droplets that appeared either dark (non-fluorescent) or exhibited red fluorescence (Figure 3f, t = t0). No exchange of the payload was observed in the dark. A brief UV light pulse (1 s) was then focused on a single TAMRA-ssDNA-containing droplet that was located close to a non-fluorescent counterpart. We were able to disassemble the target droplet without affecting the neighbouring droplets such that TAMRA-ssDNA was ejected locally into the supernatant and subsequently captured by the adjacent non-fluorescent droplet, which showed a gradual increase in red fluorescence over a period of approximately 100s (Figures 3f-h andMovie S3). From the fluorescence intensity measurements, we estimated that approximately 20% of the released ssDNA was typically captured by the neighbouring droplet depending on the diffusion distance. The locally induced chemical gradient of free TAMRA-ssDNA established immediately after UV-induced disassembly of the single droplet gave rise to an initially asymmetric distribution of the captured oligonucleotide droplet that became homogeneous with time (Figure 3f, t = t0 + 1.4s, and Figure 3g). A similar light-mediated pathway for the diffusion-limited trafficking of ssDNA was demonstrated between multiple co-located coacervate micro-droplets by sequential UV processing of individual TAMRA-ssDNA-containing droplets and neighbouring unloaded droplets (Figure S12).

In conclusion, we describe a new light-responsive DNA coacervate with photo-switchable behaviour that exhibits controllable droplet dynamics and light-activated trafficking of guest molecules. Our results highlight opportunities for controlling the spatiotemporal dynamics of DNA-containing protocells and their interplay in communities of artificial cells. For example, as DNA has been used to achieve cell-free protein expression in coacervate droplets [14] and develop programmable logic gates in synthetic protocells, [42] light-actuated DNA coacervation could be exploited to activate signalling pathways in populations of active droplets or synthetic protocells, paving the way to the construction of life-like colloidal systems capable of spatiotemporally coordinated behaviours.

Figure 1 .

 1 Figure 1. a) Scheme of the associative liquid-liquid phase separation of trans-azoTAB and dsDNA to produce coacervate droplets that are disassembled under UV light by isomerisation to cis-azoTAB and reassembled under blue light (on/off process). Programming of the reassembly process is achieved by regulating the cis-trans thermal relaxation in the dark at varying temperatures. b-d) Optical microscopy images of azoTAB (5 mM) mixed with dsDNA (5 mM nucleobase concentration) in the presence of 100 mM NaCl at pH 8 in the dark (b), under UV light (c), and under blue light after UV light exposure (d); scale bars, 10 μm. Insets show photographs of the corresponding turbid suspensions (dark or blue light), or the clear solution (UV). e) Fluorescence microscopy images of trans-azoTAB/dsDNA coacervates in the dark doped with TAMRA-ssDNA (left), and after sequential exposure to UV (centre) and then blue (right) light, showing release and re-sequestration of the oligonucleotide (false colouring to magenta). Scale bars, 10 μm. f) Plots of turbidity (black circles) and fraction of TAMRA-ssDNA free in solution (magenta circles) against time for TAMRA-ssDNA-doped trans-azoTAB/dsDNA droplets after sequential exposure to UV (purple area) and then blue (blue area) light. Error bars represent standard deviations of the average value measured on three different samples. g) Time series of optical microscopy images of a UV-adapted cis-azoTAB/dsDNA solution stored at 35°C in the dark, showing gradual nucleation and growth of trans-azoTAB/dsDNA droplets via spontaneous cis-trans thermal relaxation; scale bars, 10 μm. h) Plots showing time-dependent increase of the turbidity of UV-equilibrated cis-azoTAB/dsDNA solutions stored in the dark at varying temperatures (from 15°C (right, dark blue circles) to 60°C (left, dark red circles) in 5°C intervals).

Figure 2 .

 2 Figure 2. a) Scheme of light-activated oligonucleotide transfer from trans-azoTAB/dsDNA droplets (area 1) to photo-inactive PDDA/dsDNA droplets (area 2) across an agarose hydrogel barrier. TAMRA-ssDNA is retained within the light-responsive droplets in the dark but released on exposure to UV. Diffusive transfer of the oligonucleotides through the hydrogel gives rise to sequestration of the payload in the PDDA/dsDNA droplets. b-d) Brightfield (b) and epifluorescence (c,d) microscopy images of areas 1 and 2 in the dark (b,c) and after 210 min of UV light irradiation (d), showing transfer of TAMRA-ssDNA to the PDDA/dsDNA droplets (false colouring to magenta used). The mean grey values were adjusted between 0 and 45 on the fluorescence images of area 2 to emphasize low levels of fluorescence, while they were left between 0 and 255 on the fluorescence images of area 1. Scale bars, 20 μm.

Figure 3 .

 3 Figure 3. a) Scheme of light-activated oligonucleotide mixing and hybridization in a binary population of trans-azoTAB/dsDNA coacervate droplets containing complementary TAMRA-ssDNA or FAM-ssDNA. UV-induced release of the oligonucleotides results in FRET pairing. Subsequent exposure to blue light results in reassembly of the droplets and capture of the hybridized strands. b,c) Confocal fluorescence microscopy images of a binary population of trans-azoTAB/dsDNA droplets doped with TAMRA-ssDNA or FAM-ssDNA (false colouring to magenta and cyan, respectively), before (b) and after (c) sequential exposure to UV and blue light. Inset in c shows co-localization scatterplot (Pearson's correlation constant, ρ = 0.79)). Scale bars, 20 μm. d) Normalized FRET signal in droplets before and after sequential UV-then-blue light irradiation, showing low level of FRET signal in droplets containing only FAM-ssDNA or TAMRA-ssDNA (before light), and increased FRET signal in droplets containing both oligonucleotides (after light). Error bars represent standard deviation from the average value measured on 20 droplets in three different fields of view of the same sample. e) Scheme of UV light-activated trafficking of oligonucleotides from a single photoswitchable trans-azoTAB/dsDNA droplet doped with TAMRA-ssDNA (droplet 1) to a second non-fluorescent trans-azoTAB/dsDNA droplet devoid of oligonucleotide (droplet 2). A short (1 s) UV pulse focused on 1 results in release of the payload followed by its diffusion and sequestration within 2. f) Optical (top) and confocal fluorescence (bottom) microscopy images of two different droplets (1 and 2) before and after droplet 1 is exposed briefly to UV light (at t = t0). Selective disassembly of 1 is observed at t = t0 + 1.4 s along with the early stages of TAMRA-ssDNA transfer to 2. Inset at t = t0 + 1.4 s displays an enlarged image of droplet 2 in false colouring (colour scale from 10 to 140) showing the initial localized increase in fluorescence intensity on the side of the droplet closest to the diffusion front emanating from droplet 1. False colouring is used to emphasize low levels of fluorescence (colour scale from 0 to 255 mean grey value). Scale bars, 10 μm and 1 μm (inset). g) Time-dependent evolution of line profiles across droplet 2 (white dotted arrow at t = t0 in f); black arrow denotes edge of droplet 2 closest to the disassembling droplet 1. h) Plot of the mean fluorescence intensity at the centre of droplet 2 as a function of time before and after targeted disassembly of droplet 1 (at t = t0). The red line is a fit to a mono-exponential function.

Acknowledgements

This work was supported by the EPSRC (EP/L002957/1), the ERC Advanced Grant Scheme (EC-2016-ADG 740235), and BrisSynBio, a BBSRC/EPSRC Synthetic Biology Research Centre (BB/L01386X/1). N.M. acknowledges funding from IdEx Bordeaux (ANR-10-IDEX-03-02), a program of the French government managed by the Agence Nationale de la Recherche.

Conflict of interest

The authors declare no conflict of interest.

Keywords: azobenzene • coacervates • DNA • photochromism • protocells