Ancient DNA of crested penguins: Testing for temporal genetic shifts in the world's most diverse penguin clade.

Human impacts have substantially reduced avian biodiversity in many parts of the world, particularly on isolated islands of the Pacific Ocean. The New Zealand archipelago, including its five subantarctic island groups, holds breeding grounds for a third of the world's penguin species, including several representatives of the diverse crested penguin genus Eudyptes. While this species-rich genus has been little studied genetically, recent population estimates indicate that several Eudyptes taxa are experiencing demographic declines. Although crested penguins are currently limited to southern regions of the New Zealand archipelago, prehistoric fossil and archaeological deposits suggest a wider distribution during prehistoric times, with breeding ranges perhaps extending to the North Island. Here, we analyse ancient, historic and modern DNA sequences to explore two hypotheses regarding the recent history of Eudyptes in New Zealand, testing for (1) human-driven extinction of Eudyptes lineages; and (2) reduced genetic diversity in surviving lineages. From 83 prehistoric bone samples, each tentatively identified as 'Eudyptes spp.', we genetically identified six prehistoric penguin taxa from mainland New Zealand, including one previously undescribed genetic lineage. Moreover, our Bayesian coalescent analyses indicated that, while the range of Fiordland crested penguin (E. pachyrhynchus) may have contracted markedly over the last millennium, genetic DNA diversity within this lineage has remained relatively constant. This result contrasts with human-driven biodiversity reductions previously detected in several New Zealand coastal vertebrate taxa.

A Time-Kill Assay Study on the Synergistic Bactericidal Activity of Pomegranate Rind Extract and Zn (II) against Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa.

There is a need for new antimicrobial systems due to increased global resistance to current antimicrobials. Pomegranate rind extract (PRE) and Zn (II) ions both possess a level of antimicrobial activity and work has previously shown that PRE/Zn (II) in combination possesses synergistic activity against Herpes simplex virus and Micrococcus luteus. Here, we determined whether such synergistic activity extended to other, more pathogenic, bacteria. Reference strains of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were cultured and subjected to challenge by PRE, Zn (II), or PRE + Zn (II), in time-kill assays. Data were obtained independently by two researchers using different PRE preparations. Statistically significant synergistic activity for PRE + Zn (II) was shown for all four bacterial strains tested compared to untreated controls, although the extent of efficacy and timescales varied. Zn (II) exerted activity and at 1 h, it was not possible to distinguish with PRE + Zn (II) combination treatment in all cases. PRE alone showed low activity against all four bacteria. Reproducible synergistic bactericidal activity involving PRE and Zn (II) has been confirmed. Potential mechanisms are discussed. The development of a therapeutic system that possesses demonstrable antimicrobial activity is supported which lends itself particularly to topical delivery applications, for example MRSA infections.

High-coverage genomes to elucidate the evolution of penguins.

BACKGROUND: Penguins (Sphenisciformes) are a remarkable order of flightless wing-propelled diving seabirds distributed widely across the southern hemisphere. They share a volant common ancestor with Procellariiformes close to the Cretaceous-Paleogene boundary (66 million years ago) and subsequently lost the ability to fly but enhanced their diving capabilities. With ∼20 species among 6 genera, penguins range from the tropical Galápagos Islands to the oceanic temperate forests of New Zealand, the rocky coastlines of the sub-Antarctic islands, and the sea ice around Antarctica. To inhabit such diverse and extreme environments, penguins evolved many physiological and morphological adaptations. However, they are also highly sensitive to climate change. Therefore, penguins provide an exciting target system for understanding the evolutionary processes of speciation, adaptation, and demography. Genomic data are an emerging resource for addressing questions about such processes. RESULTS: Here we present a novel dataset of 19 high-coverage genomes that, together with 2 previously published genomes, encompass all extant penguin species. We also present a well-supported phylogeny to clarify the relationships among penguins. In contrast to recent studies, our results demonstrate that the genus Aptenodytes is basal and sister to all other extant penguin genera, providing intriguing new insights into the adaptation of penguins to Antarctica. As such, our dataset provides a novel resource for understanding the evolutionary history of penguins as a clade, as well as the fine-scale relationships of individual penguin lineages. Against this background, we introduce a major consortium of international scientists dedicated to studying these genomes. Moreover, we highlight emerging issues regarding ensuring legal and respectful indigenous consultation, particularly for genomic data originating from New Zealand Taonga species. CONCLUSIONS: We believe that our dataset and project will be important for understanding evolution, increasing cultural heritage and guiding the conservation of this iconic southern hemisphere species assemblage.

Marathon penguins - Reasons and consequences of long-range dispersal in Fiordland penguins / Tawaki during the pre-moult period.

Migratory species often roam vast distances bringing them into contact with diverse conditions and threats that could play significant roles in their population dynamics. This is especially true if long-range travels occur within crucial stages of a species' annual life-cycle. Crested penguins, for example, usually disperse over several hundreds of kilometres after completing the energetically demanding breeding season and in preparation for the costly annual moult. A basic understanding of crested penguins' pre-moult dispersal is therefore paramount in order to be able to assess factors affecting individual survival. The Fiordland penguin, or Tawaki, the only crested penguin species breeding on the New Zealand mainland, is currently one of the least studied and rarest penguin species in the world. We successfully satellite tracked the pre-moult dispersal of 17 adult Tawaki from a single colony located in the species' northern breeding distribution. Over the course of 8-10 weeks the penguins travelled up to 2,500 km away from their breeding colony, covering total swimming distances of up to 6,800 km. During outbound travels all penguins headed south-west within a well-defined corridor before branching out towards two general trip destinations. Birds leaving in late November travelled towards the Subtropical Front some 800 km south of Tasmania, whereas penguins that left in December headed further towards the subantarctic front. Using K-select analysis we examined the influence of oceanographic factors on the penguins' dispersal. Water depth, surface current velocity and sea level anomalies had the greatest influence on penguin movements at the subantarctic Front, while sea surface temperature and chlorophyll a concentration were key for birds travelling to the subtropical front. We discuss our findings in the light of anthropogenic activities (or lack thereof) in the regions visited by the penguins as well as the potential consequences of Tawaki pre-moult dispersal for the species' breeding distribution on the New Zealand mainland.

Anti-inflammatory activity of Punica granatum L. (Pomegranate) rind extracts applied topically to ex vivo skin.

Coadministered pomegranate rind extract (PRE) and zinc (II) produces a potent virucidal activity against Herpes simplex virus (HSV); however, HSV infections are also associated with localised inflammation and pain. Here, the objective was to determine the anti-inflammatory activity and relative depth penetration of PRE, total pomegranate tannins (TPT) and zinc (II) in skin, ex vivo. PRE, TPT and ZnSO4 were dosed onto freshly excised ex vivo porcine skin mounted in Franz diffusion cells and analysed for COX-2, as a marker for modulation of the arachidonic acid inflammation pathway, by Western blotting and immunohistochemistry. Tape stripping was carried out to construct relative depth profiles. Topical application of PRE to ex vivo skin downregulated expression of COX-2, which was significant after just 6h, and maintained for up to 24h. This was achieved with intact stratum corneum, proving that punicalagin penetrated skin, further supported by the depth profiling data. When PRE and ZnSO4 were applied together, statistically equal downregulation of COX-2 was observed when compared to the application of PRE alone; no effect followed the application of ZnSO4 alone. TPT downregulated COX-2 less than PRE, indicating that tannins alone may not be entirely responsible for the anti-inflammatory activity of PRE. Punicalagin was found throughout the skin, in particular the lower regions, indicating appendageal delivery as a significant route to the viable epidermis. Topical application of TPT and PRE had significant anti-inflammatory effects in ex vivo skin, confirming that PRE penetrates the skin and modulates COX-2 regulation in the viable epidermis. Pomegranates have potential as a novel approach in ameliorating the inflammation and pain associated with a range of skin conditions, including cold sores and herpetic stromal keratitis.

Correction: Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

[This corrects the article DOI: 10.1371/journal.pone.0179291.].

Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

BACKGROUND: There is a clinical need for new therapeutic products against Herpes simplex virus (HSV). The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE) and co-administered zinc (II) ions. MATERIALS AND METHODS: PRE was used in conjunction with zinc (II) salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit. RESULTS: Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 µg mL-1) a value comparable to aciclovir (EC50 = 0.18 µg mL-1); however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 µg mL-1), whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution. CONCLUSIONS: The potentiated virucidal activity of PRE by coadministered zinc (II) has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

In vitro permeation and biological activity of punicalagin and zinc (II) across skin and mucous membranes prone to Herpes simplex virus infection.

Coadministration of pomegranate rind extract (PRE) and zinc (II) ions has recently been reported as a potential new topical treatment for Herpes simplex virus (HSV) infections. In the current work we examined the in vitro topical delivery of punicalagin (major phytochemical of PRE) and zinc from hydrogels across epithelial membranes that can become infected with HSV. Porcine epidermal, buccal and vaginal mucous membranes were excised and mounted in Franz diffusion cells and dosed with a simple hydrogel containing PRE and zinc sulphate (ZnSO4). The permeation of punicalagin and zinc were determined by HPLC and ICPMS respectively; punicalagin was also determined in the basal layers by reverse tape stripping. Receptor phases from the epidermal membrane experiment were also used to challenge HSV-1 in Vero host cells, and ex vivo porcine skin was used to probe COX-2 modulation. Punicalagin and zinc permeated each of the three test membranes, with significantly greater amounts of both delivered across the epidermal membrane. The amounts of punicalagin permeating the buccal and vaginal membranes were similar, although the amount of zinc permeating the vaginal membrane was comparatively very large - it is known that zinc interacts with vaginal mucosa. The punicalagin recovered by reverse tape stripping of the epidermal, buccal and vaginal membranes gave 0.47±0.016, 0.45±0.052 and 0.51±0.048nMcm-2 respectively, and were statistically the same (p<0.05). A 2.5 log reduction was achieved against HSV-1 using diffusion cell receptor phase, and COX-2 expression was reduced by 64% in ex vivo skin after 6h. Overall, a hydrogel containing 1.25mgmL-1 PRE and 0.25M ZnSO4 was able to topically deliver both the major bioactive compound within PRE and Zn (II) across all membranes and into the site specific region of Herpes simplex vesicular clusters, while maintaining potentiated virucidal and anti-inflammatory properties. This novel therapeutic system therefore has potential for the topical treatment of HSV infections.

Quantifying climate change impacts emphasises the importance of managing regional threats in the endangered Yellow-eyed penguin.

Climate change is a global issue with effects that are difficult to manage at a regional scale. Yet more often than not climate factors are just some of multiple stressors affecting species on a population level. Non-climatic factors-especially those of anthropogenic origins-may play equally important roles with regard to impacts on species and are often more feasible to address. Here we assess the influence of climate change on population trends of the endangered Yellow-eyed penguin (Megadyptes antipodes) over the last 30 years, using a Bayesian model. Sea surface temperature (SST) proved to be the dominating factor influencing survival of both adult birds and fledglings. Increasing SST since the mid-1990s was accompanied by a reduction in survival rates and population decline. The population model showed that 33% of the variation in population numbers could be explained by SST alone, significantly increasing pressure on the penguin population. Consequently, the population becomes less resilient to non-climate related impacts, such as fisheries interactions, habitat degradation and human disturbance. However, the extent of the contribution of these factors to declining population trends is extremely difficult to assess principally due to the absence of quantifiable data, creating a discussion bias towards climate variables, and effectively distracting from non-climate factors that can be managed on a regional scale to ensure the viability of the population.

Straight line foraging in yellow-eyed penguins: new insights into cascading fisheries effects and orientation capabilities of marine predators.

Free-ranging marine predators rarely search for prey along straight lines because dynamic ocean processes usually require complex search strategies. If linear movement patterns occur they are usually associated with travelling events or migratory behaviour. However, recent fine scale tracking of flying seabirds has revealed straight-line movements while birds followed fishing vessels. Unlike flying seabirds, penguins are not known to target and follow fishing vessels. Yet yellow-eyed penguins from New Zealand often exhibit directed movement patterns while searching for prey at the seafloor, a behaviour that seems to contradict common movement ecology theories. While deploying GPS dive loggers on yellow-eyed penguins from the Otago Peninsula we found that the birds frequently followed straight lines for several kilometres with little horizontal deviation. In several cases individuals swam up and down the same line, while some of the lines were followed by more than one individual. Using a remote operated vehicle (ROV) we found a highly visible furrow on the seafloor most likely caused by an otter board of a demersal fish trawl, which ran in a straight line exactly matching the trajectory of a recent line identified from penguin tracks. We noted high abundances of benthic scavengers associated with fisheries-related bottom disturbance. While our data demonstrate the acute way-finding capabilities of benthic foraging yellow-eyed penguins, they also highlight how hidden cascading effects of coastal fisheries may alter behaviour and potentially even population dynamics of marine predators, an often overlooked fact in the examination of fisheries' impacts.

Elevated hormonal stress response and reduced reproductive output in Yellow-eyed penguins exposed to unregulated tourism.

The endangered, endemic Yellow-eyed penguin (Megadyptes antipodes) is one of the flagship species for New Zealand's wildlife tourism, and recently concern has been raised that tourism-related pressures may be becoming too great. We compared two neighbouring breeding areas exposed to different levels of human disturbance. Penguins at the site exposed to unregulated tourism showed significantly lower breeding success and fledging weights than those in an area visited infrequently for monitoring purposes only. High parental baseline corticosterone concentrations correlated with lower fledgling weights at both sites. Stress-induced corticosterone concentrations were significantly higher at the tourist-exposed site, suggesting birds have been sensitized by frequent disturbance. Consequences are likely to include reduced juvenile survival and recruitment to the tourist site, while the changed hormonal stress responses may ultimately have an effect on adult fitness and survival. For maintenance of attractive Yellow-eyed penguin-viewing destinations we recommend that tourists stay out of breeding areas and disturbance at penguin landing beaches is reduced.