Improving the success of reinforcement programs: effects of a two-week confinement in a field enclosure on the anti-predator behaviour of captive-bred European hamsters.

Captive breeding programs are an important pillar in biodiversity conservation, aiming to prevent the extinction of threatened species. However, the establishment of self-sustaining populations in the wild through the release of captive-bred animals is often hampered by a high mortality upon release. In this study, we investigated how a 2-week confinement period within a large field enclosure affected the anti-predator behaviour of 'naive' captive-bred hamsters and how potential modifications persisted over time. During three consecutive tests, hamsters were confronted with a moving predator model (a red fox mount, Vulpes vulpes) and their behaviour was filmed. After the initial round of confrontation with the predator model, one group of hamsters (field group) was released into a field enclosure protected from predators, while the other group (control) remained in their individual laboratory cages. After 2 weeks, hamsters from the field group were recaptured and individuals of both groups underwent a second confrontation test. A total of 1 month after their return from the field enclosure, field hamsters were subjected to a last confrontation test. Video analysis, investigating four behavioural variables, revealed that field hamsters significantly modified their behavioural response following the 2 weeks confinement in the enclosure, while this was not the case for control hamsters. In addition, most behavioural modifications in field hamsters persisted over 1 month, while others started to revert. We suggest that an appropriate pre-release period inside a field enclosure will enable naive (captive-bred) hamsters to develop an adequate anti-predator behaviour that will increase their immediate survival probability upon release into the wild. We believe that such measure will be of great importance for hamster conservation programs.

Stormwater ponds as an amphibian breeding site: a case study with European green toad tadpoles.

Stormwater ponds (SWPs) are built to collect and retain polluted runoff water from roads. Consequently, they are not perceived as suitable habitat for wetland species, such as many amphibians. However, given the drastic decline of wetland areas, SWPs may serve as a habitat for protected amphibian species, such as the European green toad (Bufotes viridis). The latter species is frequently found inside these artificial ponds, but their reproductive success is unknown. We assessed the suitability of SWPs as breeding habitat for European green toads by monitoring 8 SWPs and 8 semi-natural ponds (SNPs), which served as control sites. At each site, two groups of 30 tadpoles, originating at that site, were held inside two floating enclosures that contained sediment from the respective pond. During bi-weekly monitoring, tadpoles were counted and measured, allowing to estimate growth and mortality rates. A variety of biotic and abiotic factors were studied to determine the causes of potential differences in growth and mortality rates between the two pond types. While growth rate did not differ between pond types, mortality rates were significantly greater in SWPs than in SNPs. The extremely low survival rate observed in SWPs might be explained by the considerably greater pollutant concentration in their sediment and/or by the presence of leeches, which were found exclusively inside SWPs. Implementation of management measures, such as regular draining/dredging during winter, might help to lower the pollutant concentration in the sediment and reduce the density of leeches inside SWPs, improving their suitability as habitat for amphibians.

The early life of king penguins: ontogeny of dive capacity and foraging behaviour in an expert diver.

The period of emancipation in seabirds, when juveniles change from a terrestrial existence to a life at sea, is associated with many challenges. Apart from finding favourable foraging sites, they have to develop effective prey search patterns and physiological capacities that enable them to capture sufficient prey to meet their energetic needs. Animals that dive to forage, such as king penguins (Aptenodytes patagonicus), need to acquire an adequate breath-hold capacity, allowing them to locate and capture prey at depth. To investigate the ontogeny of their dive capacity and foraging performance, we implanted juvenile king penguins before their first departure to sea and also adult breeders with a data-logger recording pressure and temperature. We found that juvenile king penguins possess a remarkable dive capacity when leaving their natal colony, enabling them to conduct dives in excess of 100 m within their first week at sea. Despite this, juvenile dive/foraging performance, investigated in relation to dive depth, remained below the adult level throughout their first year at sea, probably reflecting physiological limitations as a result of incomplete maturation. A significantly shallower foraging depth of juveniles, particularly during their first 5 months at sea, could also indicate differences in foraging strategy and targeted prey. The initially greater wiggle rate suggests that juveniles fed opportunistically and also targeted different prey from adults and/or that many of the wiggles of juveniles reflect unsuccessful prey-capture attempts, indicating a lower foraging proficiency. After 5 months, this difference disappeared, suggesting sufficient physical maturation and improvement of juvenile foraging skills.

Similar circling movements observed across marine megafauna taxa.

Advances in biologging technology have enabled 3D dead-reckoning reconstruction of marine animal movements at spatiotemporal scales of meters and seconds. Examining high-resolution 3D movements of sharks (Galeocerdo cuvier, N = 4; Rhincodon typus, N = 1), sea turtles (Chelonia mydas, N = 3), penguins (Aptenodytes patagonicus, N = 6), and marine mammals (Arctocephalus gazella, N = 4; Ziphius cavirostris, N = 1), we report the discovery of circling events where animals consecutively circled more than twice at relatively constant angular speeds. Similar circling behaviors were observed across a wide variety of marine megafauna, suggesting these behaviors might serve several similar purposes across taxa including foraging, social interactions, and navigation.

The dive performance of immature king penguins following their annual molt suggests physiological constraints.

Like all birds, penguins undergo periodic molt, during which they replace old feathers. However, unlike other birds, penguins replace their entire plumage within a short period while fasting ashore. During molt, king penguins (Aptenodytes patagonicus) lose half of their initial body mass, most importantly their insulating subcutaneous fat and half of their pectoral muscle mass. The latter might challenge their capacity to generate and sustain a sufficient mechanical power output to swim to distant food sources and propel themselves to great depth for successful prey capture. To investigate the effects of the annual molt fast on their dive/foraging performance, we studied various dive/foraging parameters and peripheral temperature patterns in immature king penguins across two molt cycles, after birds had spent their first and second year at sea, using implanted data-loggers. We found that the dive/foraging performance of immature king penguins was significantly reduced during post-molt foraging trips. Dive and bottom duration for a given depth were shorter during post-molt and post-dive surface interval duration was longer, reducing overall dive efficiency and underwater foraging time. We attribute this decline to the severe physiological changes that birds undergo during their annual molt. Peripheral temperature patterns differed greatly between pre- and post-molt trips, indicating the loss of the insulating subcutaneous fat layer during molt. Peripheral perfusion, as inferred from peripheral temperature, was restricted to short periods at night during pre-molt but occurred throughout extended periods during post-molt, reflecting the need to rapidly deposit an insulating fat layer during the latter period.

Captive-reared European hamsters follow an offensive strategy during risk-assessment.

Understanding whether captive-reared animals destined to reintroduction are still able to discriminate predators has important implications for conservation biology. The endangered European hamster benefits from conservation programs throughout Europe, in which several thousand individuals are released into the wild every year. Despite this, the anti-predator strategy of hamsters and their ability to maintain predator discrimination in captivity remain to be investigated. Here, we explore the predator discrimination behaviour of captive-reared European hamsters and their response to different predation cues. When first exposed to the urine of cats and goats in a Y-maze test, hamsters spent more time close to the cat scent rather than to the goat scent. In a second experiment, during which hamsters were exposed to a non-mobile European ferret (inside a cage), hamsters significantly increased the time spent close to the ferret's cage and displayed aggressive behaviour towards the ferret. Furthermore, they did not take refuge inside an anti-predation tube (APT), a device designed to upgrade wildlife underpasses and reconnect wild hamster populations. Finally, when exposed to a mobile ferret (but without physical contact), hamsters displayed mobbing and aggressive behaviours towards the ferret, before taking refuge inside the APT. Taken together, our results show that captive-reared hamsters are still able to detect and react to predation cues, but that they initially adopt an offensive strategy (grunting, spitting, mobbing) during the risk-assessment phase. After risk assessment, however, hamsters used the APT as a refuge. Our study provides important insights into the anti-predator behaviour of hamsters. Testing the efficacy of the APT, a device that will allow upgrading wildlife underpasses for the hamster and other rodents, is also of great importance and is instrumental in conservation efforts for these species.

Why implantation of bio-loggers may improve our understanding of how animals cope within their natural environment.

Bio-loggers are miniaturized autonomous devices that record quantitative data on the state of free-ranging animals (e.g. behavior, position and physiology) and their natural environment. This is especially relevant for species where direct visual observation is difficult or impossible. Today, ongoing technical development allows the monitoring of numerous parameters in an increasing range of species over extended periods. However, the external attachment of devices might affect various aspects of animal performance (energetics, thermoregulation, foraging as well as social and reproductive behavior), which ultimately affect fitness. External attachment might also increase entanglement risk and the conspicuousness of animals, leaving them more vulnerable to predation. By contrast, implantation of devices can mitigate many of these undesirable effects and might be preferable, especially for long-term studies, provided that the many challenges associated with surgical procedures can be mastered. Implantation may then allow us to gather data that would be impossible to obtain otherwise and thereby may provide new and ecologically relevant insights into the life of wild animals. Here, we: (i) discuss the pros and cons of attachment methods; (ii) highlight recent field studies that used implanted bio-loggers to address eco-physiological questions in a wide range of species; and (iii) discuss logger implantation in light of ethical considerations.

Monocultural sowing in mesocosms decreases the species richness of weeds and invertebrates and critically reduces the fitness of the endangered European hamster.

Intensive cereal monoculture is currently the main cause of biodiversity decline in Europe. However, it is difficult to disentangle the effects of intensive monoculture (e.g. pesticide use, mechanical ploughing and reduced protective cover), let alone evaluate how far the reduction of crop diversity affects biodiversity. It remains unclear to which extent the consequent decrease in food resources affects farmland biodiversity, and particularly vertebrate species. We therefore designed this study in mesocosms to investigate the effects of monoculture crops (organic wheat or corn seeds) and mixed crops (a combination of organic wheat, corn, sunflower and alfalfa seeds) on (1) the species richness of weeds and invertebrates and (2) the reproductive success of the European hamster (Cricetus cricetus), a critically endangered umbrella species of European farmlands. We found a negative impact of organic monoculture crops on plant and invertebrate species richness, with values respectively 38% and 28% lower than those obtained for mixed organic crops. The reproductive success of hamsters was reduced by 82% in monoculture mesocosms. These results highlight that monoculture per se can be detrimental for farmland biodiversity (i.e. from plants to vertebrates), even before taking into account the use of pesticide and mechanization. We believe that future research should further consider how food reduction in agroecosystems affects farmland wildlife, including vertebrates. Moreover, we argue that conservation actions must focus on restoring plant diversity on farmland to reverse the observed trend in farmland wildlife decline.

Thermal strategies of king penguins during prolonged fasting in water.

Most animals experience periods of unfavourable conditions, challenging their daily energy balance. During breeding, king penguins fast voluntarily for up to 1.5 months in the colony, after which they replenish their energy stores at sea. However, at sea, birds might encounter periods of low foraging profitability, forcing them to draw from previously stored energy (e.g. subcutaneous fat). Accessing peripheral fat stores requires perfusion, increasing heat loss and thermoregulatory costs. Hence, how these birds balance the conflicting demands of nutritional needs and thermoregulation is unclear. We investigated the physiological responses of king penguins to fasting in cold water by: (1) monitoring tissue temperatures, as a proxy of tissue perfusion, at four distinct sites (deep and peripheral); and (2) recording their oxygen consumption rate while birds floated inside a water tank. Despite frequent oscillations, temperatures of all tissues often reached near-normothermic levels, indicating that birds maintained perfusion to peripheral tissues throughout their fasting period in water. The oxygen consumption rate of birds increased with fasting duration in water, while it was also higher when the flank tissue was warmer, indicating greater perfusion. Hence, fasting king penguins in water maintained peripheral perfusion, despite the associated greater heat loss and, therefore, thermoregulatory costs, probably to access subcutaneous fat stores. Hence, the observed normothermia in peripheral tissues of king penguins at sea, upon completion of a foraging bout, is likely explained by their nutritional needs: depositing free fatty acids (FFA) in subcutaneous tissues after profitable foraging or mobilizing FFA to fuel metabolism when foraging success was insufficient.

A comparison of camera trap and permanent recording video camera efficiency in wildlife underpasses.

In the current context of biodiversity loss through habitat fragmentation, the effectiveness of wildlife crossings, installed at great expense as compensatory measures, is of vital importance for ecological and socio-economic actors. The evaluation of these structures is directly impacted by the efficiency of monitoring tools (camera traps…), which are used to assess the effectiveness of these crossings by observing the animals that use them. The aim of this study was to quantify the efficiency of camera traps in a wildlife crossing evaluation. Six permanent recording video systems sharing the same field of view as six Reconyx HC600 camera traps installed in three wildlife underpasses were used to assess the exact proportion of missed events (event being the presence of an animal within the field of view), and the error rate concerning underpass crossing behavior (defined as either Entry or Refusal). A sequence of photographs was triggered by either animals (true trigger) or artefacts (false trigger). We quantified the number of false triggers that had actually been caused by animals that were not visible on the images ("false" false triggers). Camera traps failed to record 43.6% of small mammal events (voles, mice, shrews, etc.) and 17% of medium-sized mammal events. The type of crossing behavior (Entry or Refusal) was incorrectly assessed in 40.1% of events, with a higher error rate for entries than for refusals. Among the 3.8% of false triggers, 85% of them were "false" false triggers. This study indicates a global underestimation of the effectiveness of wildlife crossings for small mammals. Means to improve the efficiency are discussed.

Apparent changes in body insulation of juvenile king penguins suggest an energetic challenge during their early life at sea.

Little is known about the early life at sea of marine top predators, like deep-diving king penguins (Aptenodytes patagonicus), although this dispersal phase is probably a critical phase in their life. Apart from finding favourable foraging sites, they have to develop effective prey search patterns as well as physiological capacities that enable them to capture sufficient prey to meet their energetic needs. To investigate the ontogeny of their thermoregulatory responses at sea, we implanted 30 juvenile king penguins and 8 adult breeders with a small data logger that recorded pressure and subcutaneous temperature continuously for up to 2.5 years. We found important changes in the development of peripheral temperature patterns of foraging juvenile king penguins throughout their first year at sea. Peripheral temperature during foraging bouts fell to increasingly lower levels during the first 6 months at sea, after which it stabilized. Most importantly, these changes re-occurred during their second year at sea, after birds had fasted for ∼4 weeks on land during their second moult. Furthermore, similar peripheral temperature patterns were also present in adult birds during foraging trips throughout their breeding cycle. We suggest that rather than being a simple consequence of concurrent changes in dive effort or an indication of a physiological maturation process, these seasonal temperature changes mainly reflect differences in thermal insulation. Heat loss estimates for juveniles at sea were initially high but declined to approximately half after ∼6 months at sea, suggesting that juvenile king penguins face a strong energetic challenge during their early oceanic existence.

High peripheral temperatures in king penguins while resting at sea: thermoregulation versus fat deposition.

Marine endotherms living in cold water face an energetically challenging situation. Unless properly insulated, these animals will lose heat rapidly. The field metabolic rate of king penguins at sea is about twice that on land. However, when at sea, their metabolic rate is higher during extended resting periods at the surface than during foraging, when birds descend to great depth in pursuit of their prey. This is most likely explained by differences in thermal status. During foraging, peripheral vasoconstriction leads to a hypothermic shell, which is rewarmed during extended resting bouts at the surface. Maintaining peripheral perfusion during rest in cold water, however, will greatly increase heat loss and, therefore, thermoregulatory costs. Two hypotheses have been proposed to explain the maintenance of a normothermic shell during surface rest: (1) to help the unloading of N2 accumulated during diving; and (2) to allow the storage of fat in subcutaneous tissue, following the digestion of food. We tested the latter hypothesis by maintaining king penguins within a shallow seawater tank, while we recorded tissue temperature at four distinct sites. When king penguins were released into the tank during the day, their body temperature immediately declined. However, during the night, periodic rewarming of abdominal and peripheral tissues occurred, mimicking temperature patterns observed in the wild. Body temperatures, particularly in the flank, also depended on body condition and were higher in 'lean' birds (after 10 days of fasting) than in 'fat' birds. While not explicitly tested, our observation that nocturnal rewarming persists in the absence of diving activity during the day does not support the N2 unloading hypothesis. Rather, differences in temperature changes throughout the day and night, and the effect of body condition/mass supports the hypothesis that tissue perfusion during rest is required for nutritional needs.

Diets derived from maize monoculture cause maternal infanticides in the endangered European hamster due to a vitamin B3 deficiency.

From 1735 to 1940, maize-based diets led to the death of hundreds of thousands of people from pellagra, a complex disease caused by tryptophan and vitamin B3 deficiencies. The current cereal monoculture trend restricts farmland animals to similarly monotonous diets. However, few studies have distinguished the effects of crop nutritional properties on the reproduction of these species from those of other detrimental factors such as pesticide toxicity or agricultural ploughing. This study shows that maize-based diets cause high rates of maternal infanticides in the European hamster, a farmland species on the verge of extinction in Western Europe. Vitamin B3 supplementation is shown to effectively restore reproductive success in maize-fed females. This study pinpoints how nutritional deficiencies caused by maize monoculture could affect farmland animal reproduction and hence their fitness.

Associations between Resting, Activity, and Daily Metabolic Rate in Free-Living Endotherms: No Universal Rule in Birds and Mammals.

Energy management models provide theories and predictions for how animals manage their energy budgets within their energetic constraints, in terms of their resting metabolic rate (RMR) and daily energy expenditure (DEE). Thus, uncovering what associations exist between DEE and RMR is key to testing these models. Accordingly, there is considerable interest in the relationship between DEE and RMR at both inter- and intraspecific levels. Interpretation of the evidence for particular energy management models is enhanced by also considering the energy spent specifically on costly activities (activity energy expenditure [AEE] = DEE - RMR). However, to date there have been few intraspecific studies investigating such patterns. Our aim was to determine whether there is a generality of intraspecific relationships among RMR, DEE, and AEE using long-term data sets for bird and mammal species. For mammals, we use minimum heart rate (fH), mean fH, and activity fH as qualitative proxies for RMR, DEE, and AEE, respectively. For the birds, we take advantage of calibration equations to convert fH into rate of oxygen consumption in order to provide quantitative proxies for RMR, DEE, and AEE. For all 11 species, the DEE proxy was significantly positively correlated with the RMR proxy. There was also evidence of a significant positive correlation between AEE and RMR in all four mammal species but only in some of the bird species. Our results indicate there is no universal rule for birds and mammals governing the relationships among RMR, AEE, and DEE. Furthermore, they suggest that birds tend to have a different strategy for managing their energy budgets from those of mammals and that there are also differences in strategy between bird species. Future work in laboratory settings or highly controlled field settings can tease out the environmental and physiological processes contributing to variation in energy management strategies exhibited by different species.

How maize monoculture and increasing winter rainfall have brought the hibernating European hamster to the verge of extinction.

Over the last decades, climate change and agricultural intensification have been identified as two major phenomena negatively affecting biodiversity. However, little is known about their effects on the life-history traits of hibernating species living in agro-ecosystems. The European hamster (Cricetus cricetus), once a common rodent on agricultural land, is now on the verge of extinction in France. Despite the implemented measures for its protection, populations are still in sharp decline but the reasons for it remain unclear. To investigate how environmental change has affected this hibernating rodent, we used a data set based on 1468 recordings of hamster body mass at emergence from hibernation from 1937 to 2014. We reveal the adverse effects of increasing winter rainfall and maize monoculture intensification on the body mass of wild hamsters. Given the links that exist between body mass, reproductive success and population dynamics in mammals, these results are of particular importance to understand the decline of this species. In view of the rates of maize monoculture intensification and the predicted increase in winter rainfall, it is of the utmost importance to improve land management in Western Europe to avoid the extinction of this species.

Fat King Penguins Are Less Steady on Their Feet.

Returning to the shore after a feeding sojourn at sea, king penguins often undertake a relatively long terrestrial journey to the breeding colony carrying a heavy, mostly frontal, accumulation of fat along with food in the stomach for chick-provisioning. There they must survive a fasting period of up to a month in duration, during which their complete reliance on endogenous energy stores results in a dramatic loss in body mass. Our aim was to determine if the king penguin's walking gait changes with variations in body mass. We investigated this by walking king penguins on a treadmill while instrumented with an acceleration data logger. The stride frequency, dynamic body acceleration (DBA) and posture of fat (pre-fasting; 13.2 kg) and slim (post fasting; 11 kg) king penguins were assessed while they walked at the same speed (1.4 km/h) on a treadmill. Paired statistical tests indicated no evidence for a difference in dynamic body acceleration or stride frequency between the two body masses however there was substantially less variability in both leaning angle and the leaning amplitude of the body when the birds were slimmer. Furthermore, there was some evidence that the slimmer birds exhibited a decrease in waddling amplitude. We suggest the increase in variability of both leaning angle and amplitude, as well as a possibly greater variability in the waddling amplitude, is likely to result from the frontal fat accumulation when the birds are heavier, which may move the centre of mass anteriorly, resulting in a less stable upright posture. This study is the first to use accelerometry to better understand the gait of a species within a specific ecological context: the considerable body mass change exhibited by king penguins.

Large-scale climatic anomalies affect marine predator foraging behaviour and demography.

Determining the links between the behavioural and population responses of wild species to environmental variations is critical for understanding the impact of climate variability on ecosystems. Using long-term data sets, we show how large-scale climatic anomalies in the Southern Hemisphere affect the foraging behaviour and population dynamics of a key marine predator, the king penguin. When large-scale subtropical dipole events occur simultaneously in both subtropical Southern Indian and Atlantic Oceans, they generate tropical anomalies that shift the foraging zone southward. Consequently the distances that penguins foraged from the colony and their feeding depths increased and the population size decreased. This represents an example of a robust and fast impact of large-scale climatic anomalies affecting a marine predator through changes in its at-sea behaviour and demography, despite lack of information on prey availability. Our results highlight a possible behavioural mechanism through which climate variability may affect population processes.

Effect of walking speed on the gait of king penguins: An accelerometric approach.

Little is known about non-human bipedal gaits. This is probably due to the fact that most large animals are quadrupedal and that non-human bipedal animals are mostly birds, whose primary form of locomotion is flight. Very little research has been conducted on penguin pedestrian locomotion with the focus instead on their associated high energy expenditure. In animals, tri-axial accelerometers are frequently used to estimate physiological energy cost, as well as to define the behaviour pattern of a species, or the kinematics of swimming. In this study, we showed how an accelerometer-based technique could be used to determine the biomechanical characteristics of pedestrian locomotion. Eight king penguins, which represent the only family of birds to have an upright bipedal gait, were trained to walk on a treadmill. The trunk tri-axial accelerations were recorded while the bird was walking at four different speeds (1.0, 1.2, 1.4 and 1.6km/h), enabling the amplitude of dynamic body acceleration along the three axes (amplitude of DBAx, DBAy and DBAz), stride frequency, waddling and leaning amplitude, as well as the leaning angle to be defined. The magnitude of the measured variables showed a significant increase with increasing speed, apart from the backwards angle of lean, which decreased with increasing speed. The variability of the measured variables also showed a significant increase with speed apart from the DBAz amplitude, the waddling amplitude, and the leaning angle, where no significant effect of the walking speed was found. This paper is the first approach to describe 3D biomechanics with an accelerometer on wild animals, demonstrating the potential of this technique.

Reassessment of the cardio-respiratory stress response, using the king penguin as a model.

Research in to short-term cardio-respiratory changes in animals in reaction to a psychological stressor typically describes increases in rate of oxygen consumption (V̇(O2)) and heart rate. Consequently, the broad consensus is that they represent a fundamental stressor response generalizable across adult species. However, movement levels can also change in the presence of a stressor, yet studies have not accounted for this possible confound on heart rate. Thus the direct effects of psychological stressors on the cardio-respiratory system are not resolved. We used an innovative experimental design employing accelerometers attached to king penguins (Aptenodytes patagonicus) to measure and thus account for movement levels in a sedentary yet free-to-move animal model during a repeated measures stress experiment. As with previous studies on other species, incubating king penguins (N = 6) exhibited significant increases in both V̇(O2) and heart rate when exposed to the stressor. However, movement levels, while still low, also increased in response to the stressor. Once this was accounted for by comparing periods of time during the control and stress conditions when movement levels were similar as recorded by the accelerometers, only V̇(O2) significantly increased; there was no change in heart rate. These findings offer evidence that changing movement levels have an important effect on the measured stress response and that the cardio-respiratory response per se to a psychological stressor (i.e. the response as a result of physiological changes directly attributable to the stressor) is an increase in V̇(O2) without an increase in heart rate.

First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli).

The hard tick Ixodes uriae parasitises a wide range of seabird species in the circumpolar areas of both Northern and Southern hemispheres and has been shown to be infected with Borrelia burgdorferi sensu lato, the bacterial agents of Lyme borreliosis. Although it is assumed that seabirds represent viable reservoir hosts, direct demonstrations of infection are limited to a single study from the Northern hemisphere. Here, the blood of 50 tick-infested adult king penguins (Aptenodytes patagonicus halli) breeding in the Crozet Archipelago (Southern Indian Ocean) was examined for B. burgdorferi sl exposure by serology and for spirochetemia by in vitro DNA amplification. Four birds were found positive by serology, whereas B. burgdorferi sl DNA was detected in two other birds. Our data therefore provide the first direct proof of Borrelia burgdorferi sl spirochetes in seabirds of the Southern hemisphere and indicate a possible reservoir role for king penguins in the natural maintenance of this bacterium. Although the bacterial genetic diversity present in these hosts and the infectious period for tick vectors remain to be elucidated, our results add to a growing body of knowledge on the contribution of seabirds to the complex epizootiology of Lyme disease and the global dissemination of B. burgdorferi sl spirochetes.