Spheniscus Humboldti Research Articles

Diversity of MHC class I alleles in Spheniscus humboldti

The major histocompatibility complex locus (MHC) is a gene region related to immune response and exhibits a remarkably great diversity. We deduced that polymorphisms in MHC genes would help to solve several issues on penguins, including classification, phylogenetic relationship, and conservation. This study aimed to elucidate the structure and diversity of the so far unknown MHC class I gene in a penguin species. The structure of an MHC class I gene from the Humboldt penguin (Spheniscus humboldti) was determined by using an inverse PCR method. We designed PCR primers to directly determine nucleotide sequences of PCR products from the MHC class I gene and to obtain recombinant clones for investigating the diversity of the MHC class I gene in Humboldt penguins. A total of 24 MHC class I allele sequences were obtained from 40 individuals. Polymorphisms were mainly found in exons 2 and 3, as expected from the nature of MHC class I genes in vertebrate species including birds and mammals. Phylogenetic analyses of MHC class I alleles have revealed that the Humboldt penguin is closely related to the Red Knot (Calidris canutus) belonging to Charadriiformes.

БІОМОРФОЛОГІЧНІ ОСОБЛИВОСТІ СКЕЛЕТНИХ ЕЛЕМЕНТІВ ПЛЕЧОВОГО СУГЛОБА ПІНГВІНА ГУМБАЛЬТА

The article presents the results and analysis of osteometric research of skeletal structures of the shoulder joint of Humboldt penguin (Spheniscus humboldti). Statistical analysis of the obtained results was made and the percentage ratio of the skeletal structures of the shoulder joint of Humboldt penguin was determined. Certain species features and natural laws were established that point out specific biomorphological adaptations to the «underwater flight». In particular clavicles that accrete into a «fork» are not attached to the sternum. This allowed us to understand certain patterns of their development.The shoulder joint of birds is a complex joint that is formed by bones of shoulder girdle and humerus. A coracoid is the most developed bone of the shoulder girdle. It should be noted that coracoid of birds is characterized by a high degree of development, which is caused by the development of muscles of wing that are fixed to it. The proximal end of coracoid forms a so-called acro-coracoidal process, to which a collarbone is fixed. The distal end of coracoid is connected to sternum, thus forming a coraco-sternal joint that is characterized by low motility.It was found that flipper form of thoracic limb and certain structural features of skeletal structures of the shoulder joint of Sphenisciformes, in particular flattened humerus, caudaly extended scapula, powerful coracoid and furcula are apparently caused by the influence of increased functional loads that have arisen as a result of adaptation to perform rowing functions by wing during rapid and deep swimming under water, because movement under water requires more physical activity.Penguins (Humboldt penguin) are characterized by greatly elongated and expanded in its caudal part scapula (Annex A-3, Fig. 3.5). Its length relatively to the length of the humerus is 150%, and in the widest part - more than 20% of the scapula length. It should be noted that in caudal part of scapula is small, not ossified area that is covered with connective tissue membrane. In studied species acromion of scapula is directed upwards. Furcula in penguin is much expanded in its proximal part. Coracoid is like a column and elongated. Its length relatively to the length of the humerus is 111.1%. Underlying of coracoid is relatively narrow. Width of the coracoid base relatively to the lengths is 29.1%. On the lateral edge of coracoid base is a small appendage, directed proximally. In addition, the lateral edges of coracoid in studied bird species has so-called coracoid window. This window is formed as a result of coracoid ligament ossification. The proximal end of coracoid is bended laterally. Humerus in Humboldt penguin has a flattened shape. Its length relatively to the total length of the wing is 33.5%. Pneumatic fossa is large enough.

Husbandry and enclosure influences on penguin behavior and conservation breeding

Multi-zoo comparisons of animal welfare are rare, and yet vital for ensuring continued improvement of zoo enclosures and husbandry. Methods are not standardized for the development of zoo enclosures based on multiple indicators, and case study species are required. This study compares behavior and breeding success to various enclosure and husbandry parameters for the Humboldt penguin, Spheniscus humboldti, for the development of improved enclosure design. Behavioral sampling was completed at Flamingo Land over a period of 8 months. Further data on behavior, enclosure design, and breeding success were collected via questionnaires, visits to zoos, and literature review. Breeding success was primarily influenced by colony age and number of breeding pairs, suggesting an important social influence on reproduction. Across zoos, there was also significant variation in behavior. The proportion of time spent in water varied between zoos (2-23%) and was used as an indicator of physical activity and natural behavior. Regression models revealed that water-use was best predicted by total enclosure area per penguin, followed by land area, with some evidence for positive influence of pool surface area per penguin. Predominantly linear/curvilinear increases in our biological indicators with enclosure parameters suggest that optimal conditions for S. humboldti were not met among the selected zoos. We propose revised minimum conditions for S. humboldti enclosure design, which exceed those in the existing husbandry guidelines. We present a framework for the evaluation of zoo enclosures and suggest that a rigorous scientific protocol be established for the design of new enclosures, based on multivariate methods. Zoo Biol. 35:385-397, 2016. © Wiley Periodicals, Inc.

Post-moult movements of sympatrically breeding Humboldt and Magellanic Penguins in south-central Chile

Ten Humboldt (Spheniscus humboldti) and eight Magellanic Penguins (S. magellanicus) were successfully equipped with satellite transmitters in March 2009 on Islotes Puñihuil in central south-Chile to follow their post-moult dispersal. Overall, Humboldt Penguins could be followed for a mean period of 49 ±18 days (range: 25–93) and Magellanic Penguins for 57 ±12 days (range 35–68). Irrespective of species and sex, seven study birds remained in the vicinity of their breeding ground throughout the transmission period. All other penguins moved northwards, either only a relatively short distance (max 400 km) to Isla Mocha at 38°S (n=3) or further north beyond 35°S (n=8). However, eight of these birds (73%) turned south again towards the end of the individual tracking periods. The total area used by both species during the tracking period was restricted to a coastal area stretching from the breeding site at 42°S about 1000 km to the north at about 32°S. The area used by Humboldt penguins overlapped by 95% the area used by Magellanic penguins, whereas the area used by the latter species was much larger and overlapped only by 45% with the area used by Humboldt penguins. Overall, our results indicate that Magellanic Penguins in the Pacific Ocean are probably less migratory than their conspecifics on the Atlantic side, while Humboldt Penguins appear to be more migratory than previously anticipated. In general, there was a poor relationship between preferred foraging areas and chlorophyll-a, as a proxy for primary productivity, indicating the limitations of using remote-sensed primary productivity as a proxy to interpret the foraging behaviour of marine predators. In addition, there was also no clear relationship between the preferred foraging areas and the amount of regional fish catches by artisanal fishery.

The role of information in changing tourists behavioral preferences at the Humboldt penguin reserve in northern Chile.

With considerable focus on ecotourism's potential to contribute to conservation, it is increasingly important to understand the implications of ecological information in triggering sustainability-relevant attitudes and actions. This study assesses whether people who have ecological information regarding the negative impact of their recreational behavior on penguins' stress will choose to remain farther away from the penguins to avoid that impact although this option will reduce the personal benefits of their tourism experience. To answer this question, we use a choice experiment with three attributes related to “Humboldt penguin watching”: (1) price of the experience, (2) distance at which penguins could be observed, and (3) penguin density. In addition, we used two treatments: with and without ecological information. We used a pooled data (with and without information) mixed logit model to identify the effect of providing or not providing information. Using a chi-square test, we first tested whether people in the sample with information chose different alternatives than those individuals without information. Furthermore, we evaluate whether the coefficient associated with the attributes of the mixed logit model, and therefore people's behavioral preferences, differs among samples. Results show that, irrespective of socio-demographic differences, visitors with information were more prone to select alternatives that reduce penguin stress, despite more educated, wealthier, and older people tend to increase their welfare when they choose being closer to the penguins. People without information never choose the alternative which results in a reduction of penguin stress. Ecological information is shown to reverse this trend, in fact, tourists perceived (on average) a welfare loss of CL$1099 (US$1.9) if he/she is too close to the penguins once information has been granted. These results are encouraging because they support the claim that well-defined educational and informational campaigns can have important effects on the way in which people behave in areas of interest for conservation. Granting ecological information can become an important tool to encourage conservation behavior, particularly in areas where support for enforcement is weak.

Vocal individuality and species divergence in the contact calls of banded penguins

Penguins produce contact calls to maintain social relationships and group cohesion. Such vocalisations have recently been demonstrated to encode individual identity information in the African penguin. Using a source-filter theory approach, we investigated whether acoustic cues of individuality can also be found in other Spheniscus penguins and the acoustic features of contact calls have diverged within this genus. We recorded vocalisations from two ex-situ colonies of Humboldt penguin and Magellanic penguin (sympatric and potentially interbreeding in the wild) and one ex-situ group of African penguins (allopatric although capable of interbreeding with the other two species in captivity). We measured 14 acoustic parameters from each vocalisation. These included temporal (duration), source-related (fundamental frequency, f0), and filter-related (formants) parameters. They were then used to carry out a series of stepwise discriminant function analyses (with cross-validation) and General Linear Model comparisons. We showed that contact calls allow individual discrimination in two additional species of the genus Spheniscus. We also found that calls can be classified according to species in a manner far greater than that attributable by chance, even though there is limited genetic distance among African, Humboldt, and Magellanic penguins. Our results provide further evidence that the source-filter theory is a valuable framework for investigating the biologically meaningful information contained in bird vocalisations. Our findings also provide novel insights into penguin vocal communication and suggest that contact calls of the penguin family are affected by selection for individuality.

Improving the re-introduction of rescued Magellanic penguins through extended health evaluation with minimal sampling volume and material

Improving the re-introduction of rescued Magellanic penguins through extended health evaluation with minimal sampling volume and material

VORICONAZOLE TOXICITY IN MULTIPLE PENGUIN SPECIES.

Aspergillosis is a common respiratory fungal disease in penguins managed under human care. Triazole antifungal drugs, including itraconazole, are most commonly used for treatment; however, itraconazole treatment failures from drug resistance are becoming more common, requiring newer treatment options. Voriconazole, a newer triazole, is being used more often. Until recently, no voriconazole pharmacokinetic studies had been performed in penguins, leading to empiric dosing based on other avian studies. This has led to increased anecdotal reporting of apparent voriconazole toxicity in penguins. This report describes 18 probable and 6 suspected cases of voriconazole toxicity in six penguin species from nine institutions: 12 African penguins (Spheniscus demersus), 5 Humboldt penguins (Spheniscus humboldti), 3 Magellanic penguins (Spheniscus magellanicus), 2 gentoo penguins (Pygoscelis papua papua), 1 macaroni penguin (Eudyptes chrysolophus), and 1 emperor penguin (Aptenodytes forsteri). Observed clinical signs of toxicity included anorexia, lethargy, weakness, ataxia, paresis, apparent vision changes, seizure-like activity, and generalized seizures. Similar signs of toxicity have also been reported in humans, in whom voriconazole therapeutic plasma concentration for Aspergillus spp. infections is 2-6 μg/ml. Plasma voriconazole concentrations were measured in 18 samples from penguins showing clinical signs suggestive of voriconazole toxicity. The concentrations ranged from 8.12 to 64.17 μg/ml, with penguins having plasma concentrations above 30 μg/ml exhibiting moderate to severe neurologic signs, including ataxia, paresis, and seizures. These concentrations were well above those known to result in central nervous system toxicity, including encephalopathy, in humans. This case series highlights the importance of species-specific dosing of voriconazole in penguins and plasma therapeutic drug monitoring. Further investigation, including pharmacokinetic studies, is warranted. The authors recommend caution in determining voriconazole dosages for use in penguin species.

Thromboelastography in Selected Avian Species.

Currently available assay methods and reagents are not optimized for evaluating avian hemostasis; therefore, assessing avian coagulopathies is challenging. Recently, thromboelastography (TEG), which measures the viscoelastic properties of blood, has been used clinically in mammalian species to diagnose and characterize hemostatic disorders. To evaluate TEG in healthy individuals of 6 avian species, we modified existing mammalian TEG protocols to allow analysis of citrated, avian whole-blood samples collected from scarlet ibis (Eudocimus ruber) (n = 13), American flamingos ( Phoenicopterus ruber ) (n = 13), helmeted Guinea fowl ( Numida meleagris ) (n = 12), Amazon parrots (Amazona species) (n = 9), Humboldt penguins ( Spheniscus humboldti ) (n = 6), and domestic chickens (n = 16). Activated partial thromboplastin time, prothrombin time, and fibrinogen were measured as a means of comparison. Regardless of the mode of activation, clot formation in the species studied was markedly delayed compared with mammals. Because of prolonged reaction time (14.7-52.7 minutes) with kaolin and diluted tissue factor, undiluted human tissue factor was used in all avian samples because it provided the shortest reaction time. Species differed significantly in reaction time (P = .007), clotting rate (P < .001), rate of clot formation (α angle; P < .001), and maximum amplitude (P < .001) values, indicating that species-specific reference intervals are necessary. Based on these results, TEG with specific reference intervals could prove useful in evaluating avian hemostatic disorders.

Metabolite Profiles of Male and Female Humboldt Penguins

We examined 185 metabolites in 30 adult Humboldt Penguins (Spheniscus humboldti) nesting at the Punta San Juan Marine Protected Area, Peru, in order to examine gender differences in metabolome profiles, particularly those involved in metabolism and energetics. The majority of the compounds identified were fatty (26% of total identified compounds), organic (19%), and amino (16%) acids. We were able to differentiate male and female penguins with 96.6% accuracy on the basis of 12 metabolites, most of which are involved in lipid and carbohydrate metabolism. These included 2-oxoglutarate, erythronic acid, GABA, mannitol, sedoheptulose 7-phosphate, and serine and six metabolites present in higher concentrations in females compared to males (2-aminoadipic acid, O-phosphorylethanolamine, glycerol 2-phosphate, glycerol 3-phosphate, pantothenic acid, and creatinine). Of these, 2-oxoglutarate and glycerol 3-phosphate were key metabolites distinguishing gender. Our results indicated that male and female Humboldt Penguins were characterized by differing metabolic states. Such differences could be important to individual and brood survival in times of environmental stress.

Unveiling an Important Humboldt Penguin (Spheniscus humboldti) Breeding Colony in Perú and the Need for Its Protection Against the Potential Impact of Guano Harvest

Abstract. Humboldt Penguins (Spheniscus humboldti) are globally Vulnerable due to the decline of the population and to a decline in the number of breeding sites in comparison to historical records. In 2010, we observed a few thousand Humboldt Penguins nesting in a surface-nest colony on Isla Santa Rosa in southern Peru. This number was unexpected because this site has never been listed as an important Humboldt Penguin breeding ground. We visited this island again in June 2011 and 2012, and counted 1,965 and 1,745 active nests, respectively. These numbers indicate not only the presence of at least 3,500–4,000 Humboldt Penguins, but places Isla Santa Rosa among the five largest Humboldt Penguin colonies within its entire range. The need for recognition of Isla Santa Rosa as a major Humboldt Penguin refuge is crucial for the conservation of the species, as the island has remained untouched from guano harvesting since 1996. This has resulted in a significant accumulation of guano likely to be legally collecte...

The glucocorticoid stress response in Magellanic Penguins (Spheniscus magellanicus): comparing within and between breeding seasons, by age and colony, after fighting, and with other penguin species

Comparing baseline and stress-induced glucocorticoid hormone levels in animals is a popular tool to assess differences in stress experienced among groups. We compare corticosterone levels in Magellanic Penguins (Spheniscus magellanicus (J.R. Forster, 1781)) in situations where we hypothesize differences in stress patterns may exist. We compared penguins both within and between breeding seasons, birds breeding at two different locations, penguins at three different ages, penguins that had shown evidence of recent fighting, and, finally, how stress patterns differed for four different penguin species. Throughout a breeding season (settlement, incubation, and chick-rearing), we found no differences in either baseline or stress-induced hormone concentrations. Chick, juvenile, and adult penguins did not differ in baseline corticosterone levels, but juveniles had a reduced stress response. Penguins in one season showed a stress response to capture, likely due to a recent severe weather event. We found no differences in baseline or stress-induced hormone titers for birds from two different breeding locations. Evidence of recent fighting also did not alter baseline corticosterone levels. Finally, among four species of penguins measured (Magellanic, Humboldt (Spheniscus humboldti Meyen, 1834), Galápagos (Spheniscus mendiculus Sundevall, 1871), and Rockhopper (Eudyptes chrysocome (J.R. Forster, 1781)), baseline levels were always consistent, while stress-induced levels were significantly higher in the Rockhopper Penguin only.

King penguins can detect two odours associated with conspecifics.

Recent studies on olfaction in penguins have focused on their use of odours while foraging. It has been proposed for some seabirds that an olfactory landscape shaped by odours coming from feeding areas exists. Islands and colonies, however, may also contribute to the olfactory landscape and may act as an orienting map. To test sensitivities to a colony scent we studied whether King penguins (Aptenodytes patagonicus) could detect the smell of sand, feathers or feces by holding presentations beneath their beaks while they naturally slept on the beach. Penguins had a significantly greater response to the feathers and feces presentations than to sand. Although only a first step in exploring a broader role of olfaction in this species, our results raise the possibility of olfaction being used by King penguins in three potential ways: (1) locating the colony from the water or the shore, (2) finding the rendezvous zone within the colony where a chick or partner may be found, or (3) recognizing individuals by scent, as in Humboldt penguins (Spheniscus demersus).

Mortalität bei Humboldtpinguinen (Spheniscus humboldti Meyen, 1834) im Zoo Dresden – ein multifaktorielles Geschehen?

Within a four-year period, 42 Humboldt penguins (Spheniscus humboldti Meyen, 1834) kept at Zoo Dresden died from infections with different opportunistic bacterial and fungal pathogens. Despite rigorous search for underlying causes, including post mortem examinations, sample collection from living penguins and thorough inspection of the artificial environment inhabited by the birds, no single causative factor could be identified to be responsible for the highly increased mortality. Instead, the authors assume a multifactorial background. Contributing factors might include primary or predisposing infectious agents, toxic substances, stress, pool water and air quality as well as hygienic aspects like handling and preparation of fish.

Molecular Epidemiology of Avian Malaria in Wild Breeding Colonies of Humboldt and Magellanic Penguins in South America.

Avian malaria is a disease caused by species of the genera Haemoproteus, Leucocytozoon, and Plasmodium. It affects hundreds of bird species, causing varied clinical signs depending on the susceptibility of the host species. Although high mortality has been reported in captive penguins, limited epidemiological studies have been conducted in wild colonies, and isolated records of avian malaria have been reported mostly from individuals referred to rehabilitation centers. For this epidemiological study, we obtained blood samples from 501 adult Humboldt and 360 adult Magellanic penguins from 13 colonies throughout South America. To identify malaria parasitaemia, we amplified the mtDNA cytochrome b for all three parasite genera. Avian malaria was absent in most of the analyzed colonies, with exception of the Punta San Juan Humboldt penguin colony, in Peru, where we detected at least two new Haemoproteus lineages in three positive samples, resulting in a prevalence of 0.6% for the species. The low prevalence of avian malaria detected in wild penguins could be due to two possible causes: A low incidence, with high morbidity and mortality in wild penguins or alternatively, penguins sampled in the chronic stage of the disease (during which parasitaemia in peripheral blood samples is unlikely) would be detected as false negatives.

Partial cloning of CYP2C23a genes and hepatic protein expression in eight representative avian species.

Large interspecies differences in avian xenobiotic metabolism have been revealed by microsome-based studies, but specific enzyme isoforms in different bird species have not yet been compared. We have previously shown that CYP2C23 genes are the most induced CYP isoforms in chicken liver. In this study, we collected partial CYP2C23a gene sequences from eight avian species (ostrich, blue-eared pheasant, snowy owl, great-horned owl, Chilean flamingo, peregrin falcon, Humboldt penguin, and black-crowned night heron) selected to cover the whole avian lineage: Paleognathae, Galloanserae, and Neoaves. Genetic analysis showed that CYP2C23 genes of Galloanserae species (chicken and blue-eared pheasant) had unique characteristics. We found some duplicated genes (CYP2C23a and CYP2C23b) and two missing amino acid residues in Galloanserae compared to the other two lineages. The genes have lower homology than in other avian lineages, which suggests Galloanserae-specific rapid evolutionary changes. These genetic features suggested that the Galloanserae are not the most representative avian species, considering that the Neoaves comprise more than 95% of birds. Moreover, we succeeded in synthesizing an antipeptide polyclonal antibody against the region of CYP2C23 protein conserved in avians. However, comparative quantitation of CYP2C23 proteins in livers from six species showed that expression levels of these proteins differed no more than fourfold. Further study is needed to clarify the function of avian CYP2C23 proteins.

Malignant melanoma in the penguin: characterization of the clinical, histologic, and immunohistochemical features of malignant melanoma in 10 individuals from three species of penguin.

Malignant melanomas are aggressive neoplasms that are relatively common in penguins compared to other avian species. In this study, the clinical and pathologic characteristics of melanocytic neoplasms in five macaroni (Eudyptes chrysolophus), three rock hopper (Eudyptes chrysocome), and two Humboldt (Spheniscus humboldti) penguins are described. Tumors most commonly occurred in the skin of the foot or hock, and were seen in the subcutaneous muscle, especially near the beak/oral cavity. Gross lesions were usually heavily pigmented, becoming raised and ulcerated over time. Humboldt penguins had a unique presentation, forming variably pigmented, cornified lesions in the inguinal area. Original case materials were obtained from all but two cases, and were assessed to define the characteristics of malignancy, evaluate four immunohistochemical markers for melanoma, and look for factors useful to informing prognosis and clinical decisions. Diagnosis was made histologically, based on morphologic features and pigmentation. Though not necessary for diagnosis, PNL-2 was found to be a useful immunohistochemical marker. HMB-45 showed unreliable positive labelling and S-100, Melan-A and Ki67 were not useful. Several factors were associated with prognosis, including gross surface dimension, mitotic index, depth of neoplastic cell invasion, and degree of surface ulceration. Metastatic spread occurred to the liver, lung, adrenal gland, brain, and bone; all lesions showed positive labelling to PNL-2. The average survival after diagnosis was 7 mo, though complete surgical excision of tumors less than 2.0 cm was curative in two cases and radiation therapy prolonged survival in one penguin. The underlying pathogenesis associated with the high prevalence of melanocytic neoplasms in captive penguins could not be identified. Three different molecular methods were performed to look for viral particles and results were negative. Advanced age is the most probable associated risk factor; ultraviolet light and chlorine exposure, viral induction, and genetic predisposition were ruled out or considered unlikely.

Pollution, habitat loss, fishing, and climate change as critical threats to penguins.

Cumulative human impacts across the world's oceans are considerable. We therefore examined a single model taxonomic group, the penguins (Spheniscidae), to explore how marine species and communities might be at risk of decline or extinction in the southern hemisphere. We sought to determine the most important threats to penguins and to suggest means to mitigate these threats. Our review has relevance to other taxonomic groups in the southern hemisphere and in northern latitudes, where human impacts are greater. Our review was based on an expert assessment and literature review of all 18 penguin species; 49 scientists contributed to the process. For each penguin species, we considered their range and distribution, population trends, and main anthropogenic threats over the past approximately 250 years. These threats were harvesting adults for oil, skin, and feathers and as bait for crab and rock lobster fisheries; harvesting of eggs; terrestrial habitat degradation; marine pollution; fisheries bycatch and resource competition; environmental variability and climate change; and toxic algal poisoning and disease. Habitat loss, pollution, and fishing, all factors humans can readily mitigate, remain the primary threats for penguin species. Their future resilience to further climate change impacts will almost certainly depend on addressing current threats to existing habitat degradation on land and at sea. We suggest protection of breeding habitat, linked to the designation of appropriately scaled marine reserves, including in the High Seas, will be critical for the future conservation of penguins. However, large-scale conservation zones are not always practical or politically feasible and other ecosystem-based management methods that include spatial zoning, bycatch mitigation, and robust harvest control must be developed to maintain marine biodiversity and ensure that ecosystem functioning is maintained across a variety of scales.

Reference genes for quantitative gene expression studies in multiple avian species.

Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

Evidence of philopatry and natal dispersal in Humboldt Penguins

We report evidence of both philopatry and natal dispersal in Humboldt Penguins (Spheniscus humboldti) from a colony in central Chile. Between 1994 and 2001, we tagged 241 Humboldt Penguin chicks with subcutaneous transponder chips. Seven birds (3%) were found as adults at their natal colony: five were breeding (philopatric birds) and two were prospecting for nest-sites. Another four birds (2%) were found breeding at other colonies up to 90 km from the natal colony. Philopatric birds bred at 3.6-6.1 years old (mean ± s.d. = 4.8 ± 1 years) at nests located 5–80m from their natal nests (30 ± 25 m). Most philopatric and the prospecting birds used the same types of nests as their natal ones and we suggest that birds breeding for the first time may use cues of structural aspects of their natal nest when choosing a nesting site for the first time.