Characterization of microsatellite loci in Humboldt penguin (Spheniscus humboldti) and cross‐amplification in other penguin species

The Humboldt penguin (Spheniscus humboldti) was once very common throughout its range along the coast of Peru and Chile. Today, listed as endangered, it is crucial to gain an understanding of gene flow and levels of genetic variation between breeding colonies to protect this species effectively. We developed seven microsatellite primers to investigate gene flow and population structure among four colonies. Locus‐specific allelic diversity ranges from five to 11 alleles and observed heterozygosity ranges from 0.50 to 0.88. These markers cross‐amplify in eight penguin species over five genera.     

The first record of Dirofilaria immitis infection in a Humboldt penguin, Spheniscus humboldti

Dirofilaria immitis infection is an important parastic disease in many mammals, especially canids, but has not been reported in bird hosts. Filarial worms were isolated from the lumen of the right atrium of the heart and the connective tissue of the lung of a captive female Humboldt penguin, Spheniscus humboldti, that died at a zoo in Japan. One of these worms was observed morphologically and identified as D. immitis by features such as 4 pairs of cephalic papillae, 1 pair of cervical papillae, esophagus divided into 2 regions, 4 pairs of pre-anal papillae, 5 pairs of post-anal papillae, and unequal spicules. In addition, the partial DNA sequence (234 bp) of mitochondrial CO / gene of the filarial worm was identical to that of D. immitis. This is the first report of D. immitis infection in a bird.     

DNA sexing in Humboldt Penguins (Spheniscus humboldti) from feather samples

Humboldt Penguins (Spheniscus humboldti) show little sexual dimorphism, and although males are usually heavier and larger than females, sexing by direct observation may be difficult, especially in young subjects. In this paper we evaluate the utility of the molecular approach, for sexing impuberal Humboldt Penguins from feathers. Firstly, a PCR test was used employing primers that amplify the homologous region of the CHD-W gene, unique in female, and the CHD-Z gene, occurring in the two sexes. The analysis of the PCR products showed a band of 370 bp in males and two bands of 370 and 380 bp in females. Additionally, to confirm these results, the PCR products were digested with HaeIII and Asp700 for RFLP analysis. Male PCR products showed two bands (310 and 60 bp) after digestion with HaeIII, and a unique band (370 bp) using Asp700, while all fragments obtained from females resolved into three bands using both HaeIII (380, 310 and 60 bp) and Asp700 (370, 270 and 110 bp), confirming the previous PCR sex determination. Results from these two different DNA-based tests were in accordance, in all cases, with sexes checked by preliminary cloacoscopy. Thus, it was found that the PCR method from feather samples alone is sufficient, reliable and without any risks for a rapid sexing in Humboldt Penguin. This non-invasive sexing technique can be useful at any age to verify the sex ratio in field populations and for gender identification in ex situ conservation programs.     

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Evidence for gene flow differs from observed dispersal patterns in the Humboldt penguin, Spheniscus humboldti

The Humboldt penguin, once common throughout its range, is today listed as Vulnerable by the IUCN. Mark-recapture and telemetry studies indicate that adult Humboldt penguins are sedentary, suggesting strong genetic differentiation between colonies. We developed genotypes for 336 individuals at 12 microsatellite loci sampled at four different localities spanning the entire range of this species. Results show that long-term gene flow has occurred but appears to be affected by geographic distance as pairwise F _ST comparisons involving the colony at Punta San Juan (Peru) and the two colonies at Algarrobo (central Chile) and Puñihuil (southern Chile) are significant. Bayesian estimates of recent migration rates indicate substantial dispersal among all colonies. Despite the dramatic decline in numbers, we did not observe a bottleneck in any population. Furthermore, we did not detect a founder effect in the recently discovered colony at Puñihuil. As our indirect estimates signal strong gene flow between populations, we suggest that Humboldt penguin colonies need to be managed as a metapopulation rather than as discrete management units.     

Hematology and plasma chemistry values in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

To establish baseline hematologic and plasma biochemistry values in free-ranging Humboldt penguins (Spheniscus humboldti), heparinized blood samples were collected from 51 apparently healthy, adult Humboldt penguins residing at two colonies off the Chilean coast. Thirty samples were collected in April, 1992, from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September, 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Hematologic values measured include packed cell volume, leucocyte count, leucocyte differential, and the presence of blood parasites. Plasma biochemistry values measured include glucose, blood urea nitrogen, creatinine, uric acid, calcium, inorganic phosphorous, sodium, potassium, chloride, total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, and creatine kinase. Only the mean values for chloride and for the number of eosinophils differed significantly between the two sample groups. No blood parasites were seen. © 1995 Wiley-Liss, Inc.     

Visual pigments and oil droplets in the penguin,Spheniscus humboldti

Summary The photoreceptors of the penguin,Spheniscus humboldti, were examined using a microspectrophotometer. The cones could be divided into three classes based on their visual pigment absorbance spectra [max 403, 450 and 543 nm (Fig. 1)], and into five classes based on their visual pigment-oil droplet combination (Fig. 4). Oil droplets were of three types (Fig. 2). The rods contained a rhodopsin with max at 504 nm. No double cones were observed. The penguin should be capable of good wavelength discrimination in the blue-green region of the spectrum but with poor discrimination at longer wavelengths. It is concluded that the spectral properties of the cone types indicate that the photopic vision ofS. humboldti is adapted to the spectral qualities of its aquatic environment.     

Plasma tocopherol,retinol, and carotenoid concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

Plasma retinol and α-tocopherol concentrations were measured in heparinized blood samples collected from 51 free-ranging adult Humboldt penguins (Sphenicus humboldti) residing at two colonies off the Chilean coast. Thirty samples were collected in April 1992 from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Samples were assayed for retinol, retinyl palmitate, α-tocopherol, γ-tocopherol, lutein, β-cryptoxanthin, lycopene, α-carotene, and β-carotene. Retinol, α-tocopherol, and lutein were detected in all samples, while lycopene and γ-tocopherol were not detected in any. A significantly higher percentage of samples had detectable levels of retinyl palmitate and α-carotene in April (P < 0.001): for β-cryptoxanthin the percentage was higher in September (P < 0.001). Plasma concentrations of α-tocopherol and lutein were higher in September. Alpha-tocopherol concentrations were 1,877.1 ± 99.0 (SEM) μg/dl in April compared to 2.289 ± 122.3 μg/dl in September (P < 0.05); lutein concentrations were 4.16 ± 0.43 μg/dl in April vs. 10.68 ± 1.02 μg/dl in September (P < 0.001). Retinol concentrations were not significantly different (117 ± 8.0 μg/dl in April vs. 105.3 ± 7.6 μg/dl in September). Both physiologic changes associated with season, and the change in locale may have contributed to the differences seen in the assay means and the number of samples with detectable levels. © 1996 Wiley-Liss, Inc.     

Circulating nutrient concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Punta San Juan,Peru

Free-ranging Humboldt penguin (HP, Spheniscus humboldti) populations are under pressure from resource competition with industrial fisheries, habitat loss, and El Niño Southern Oscillation events. Foraging patterns for this top marine predator change during periods of aberrant oceanographic conditions and scarce fish stock numbers. These radical dietary fluctuations can lead to poor fertility, early embryonic death, poor hatchability, suppressed immune function, high chick mortality, and illness. To understand the variability of nutrient status in reproductive seasons, we measured select circulating nutrient concentrations (fat-soluble vitamins A, D, E, and carotenoids, fatty acids, amino acids, minerals, and electrolytes) of 105 HP at Punta San Juan, Peru during the first reproductive seasons of 2007 and 2008. We determined significant differences in nutrient status between sexes, years of sampling, and reproductive stages. Males (4.5 ± 0.38 kg) weighed more than females (4.0 ± 0.29 kg) and exhibited higher concentrations of vitamin A (0.71 ± 0.11 vs. 0.61 ± 0.12 µg/ml) and docosahexaenoic acid (6.70 ± 1.61 vs. 5.65 ± 1.59%). Males also displayed lower concentrations of β-carotene (0.01 ± 0.01 vs. 0.012 ± 0.001 µg/ml) and phosphorus (3.43 ± 0.83 vs. 4.40 ± 1.66 mg/dl). Comparison between the 2 years showed most circulating amino acid concentrations were higher in 2007. Significant differences in circulating amino acids and vitamins were also noted between different reproductive stages. These results demonstrate concentrations of nutrients can vary due to the physiological state of the animal, as well as the overall dynamics of their marine ecosystem habitat.     

The eye of the humboldt penguin, Spheniscus humboldti: visual fields and schematic optics

Construction of a schematic eye indicates that the eye of Spheniscus humboldti is aquatic in design. The lens has a power of 100 dioptres (D) while (in air) the cornea has a power of 29 D. In air, the eye is myopic (approximately 28 D) but in water it is emmetropic. Minimum pupil size would seem insufficient to allow the pupil to function as a stenopaic aperture and increase depth of focus sufficiently to overcome the eye's aerial myopia. Entry into water reduces maximum image brightness by approximately three times. In air, the maximum width of the retinal binocular field is 45 degrees and this occurs approximately 10 degrees above the line of the bill. The bill intrudes into the retinal field and binocular field width in the plane containing the bill and the optic axes is 28 degrees. The vertical extent of the binocular field is 125 degrees. In the plane containing the optic axes the cyclopean field equals 282 degrees and the optic axes diverge by 116 degrees. In this plane the mean uniocular field is 155 degrees with the temporal hemifield approximately 11 degrees larger than the nasal hemifield. Entry into water reduces the widths of the visual fields such that maximum binocular field width is only 17 degrees and the vertical extent is reduced to about 80 degrees. Binocular vision is lost in the plane of the bill, and the uniocular retinal field is reduced by 32 degrees and the cyclopean field by 36 degrees.     

Odor-based recognition of familiar and related conspecifics: a first test conducted on captive Humboldt penguins (Spheniscus humboldti)

Studies of kin recognition in birds have largely focused on parent-offspring recognition using auditory or visual discrimination. Recent studies indicate that birds use odors during social and familial interactions and possibly for mate choice, suggesting olfactory cues may mediate kin recognition as well. Here, we show that Humboldt penguins (Spheniscus humboldti), a natally philopatric species with lifetime monogamy, discriminate between familiar and unfamiliar non-kin odors (using prior association) and between unfamiliar kin and non-kin odors (using phenotype matching). Penguins preferred familiar non-kin odors, which may be associated with the recognition of nest mates and colony mates and with locating burrows at night after foraging. In tests of kin recognition, penguins preferred unfamiliar non-kin odors. Penguins may have perceived non-kin odors as novel because they did not match the birds' recognition templates. Phenotype matching is likely the primary mechanism for kin recognition within the colony to avoid inbreeding. To our knowledge this is the first study to provide evidence of odor-based kin discrimination in a bird.     

Behavioral sex differences and activity patterns of captive humboldt penguins (Spheniscus humboldti)

Three female and two male captive Humboldt penguins (Spheniscus humboldti) were observed in an enclosed exhibit over a period of 10 months to examine behavioral sex differences and to establish whether captive Humboldt penguins exhibit behavioral activity patterns. There were no statistically significant differences between the behaviors of males and females in six categories: resting, self-maintenance, locomoting, lack of visibility, agonistic behaviors, or pair-directed behaviors. Although not statistically significant, the pair-directed activities of bowing and ecstatic displaying were found to have potential use for sexual identification. Females bowing to males accounted for 77% of the observed bows. Further, males were observed performing 68% of the observed ecstatic displays, while females performed only 32%. Four behavioral phases were defined: molting, proximity, nesting, and “other.” Statistically significant behavioral differences occurred between the four phases for all ethogram behaviors with the exception of agonistic and pair-directed behaviors. Although housed under natural daylight and in an exhibit simulating their habitat in the wild, the captive birds failed to demonstrate a breeding season but bred throughout the study. However, this may have been influenced by one or more of the following factors: a ready supply of food; an air-filtered, temperature-controlled atmosphere; and the presence of an adequate number of nesting sites.     

Influence of dietary vitamins A and E on serum alpha- and gamma-tocopherols, retinol, retinyl palmitate and carotenoid concentrations in Humboldt penguins (Spheniscus humboldti)

Serum retinol, retinyl palmitate, beta-carotene, cryptoxanthin, lutein, alpha-tocopherol and gamma-tocopherol were measured in 18 captive Humboldt penguins (Spheniscus humboldti) prior to and following the removal of Columbia River (CR) smelt (Thaleichthys pacificus) from the diet. Dietary vitamin A was reduced from 59.8 to 13.5 IU g-1 (dry matter basis) when CR smelt was removed from the diet. Minimal changes were noted in dietary vitamin E. Serum samples Without-CR smelt had significantly lower circulating retinol (1.19 +/- 0.09 vs. 1.94 +/- 0.08 micrograms ml-1) and retinyl palmitate (0.033 +/- 0.012 vs. 0.105 +/- 0.004 microgram ml-1) compared to samples With-CR. The Without-CR smelt diet resulted in increased serum alpha-tocopherol from 26.4 +/- 0.94 to 39.1 +/- 3.72 micrograms ml-1. More serum samples taken Without-CR smelt had detectable levels of gamma-tocopherol than those With-CR smelt. Serum lutein was higher for the samples taken Without versus With-CR smelt. Serum cryptoxanthin did not differ. beta-Carotene was not detected. Data indicate that high levels of dietary vitamin A can affect circulating levels of retinol, retinyl palmitate and vitamin E. Thus, dietary vitamin A and the interrelationship between vitamins A and E should be considered when assessing captive penguins.     

Semen collection,characterization, and cryopreservation in a Magellanic penguin (Spheniscus magellanicus)

A cooperative method was developed for collecting semen from a Magellanic penguin. Ejaculate parameters and semen production during a breeding season were characterized. Experiments were performed to study the effect on penguin spermatozoa of two temperatures (4°C and 21°C) for short‐term storage, and two cryoprotectants (dimethylsulfoxide [DMSO] and ethylene glycol [EG]) for long‐term storage (cryopreservation). All dilutions were made using modified Beltsville Poultry Semen Extender. Sperm quality was assessed by evaluating motility and forward progression (sperm motility index [SMI]), viability, and morphology. A total of 39 ejaculates was collected over the 40‐day study period. Thirty‐eight ejaculates contained spermatozoa, but semen quality decreased toward the end of the study period. Varying levels of urate contamination were present in all ejaculates. Sperm quality parameters were similar for diluted samples held at 4°C and 21°C, and samples maintained high numbers of viable (77.8 ± 5.4%) and morphologically normal (67.9 ± 2.5%) spermatozoa at 3 hr. SMI and percentage of viable sperm decreased (P < 0.05) and the number of spermatozoa with a bent head or midpiece increased (P < 0.05) for both temperature groups over the 3‐hr storage interval. DMSO and EG were equally effective in maintaining penguin sperm quality parameters during the cryopreservation and thawing process. Frozen‐thawed semen maintained 69 ± 5 and 78 ± 3% of its pre‐freeze SMI and viability, respectively. SMI and viability decreased slightly during the cooling and equilibration phases but remained relatively stable during the 3‐hr storage interval post‐thaw. Frozen‐thawed semen also exhibited an increase (P < 0.05) in spermatozoa with a bent head or midpiece over time. The pre‐freeze SMI was higher (P < 0.05) for ejaculates with low levels of urates (clean ejaculates) compared with ejaculates with high levels of urate contamination, but sperm viability and morphology were similar (P > 0.05). Both SMI and viability of frozen‐thawed spermatozoa were higher (P < 0.05) for clean than for contaminated ejaculates. This is the first report on penguin ejaculate parameters, semen production, and preliminary methods for short‐ and long‐term semen storage. Zoo Biol 18:199–214, 1999. © 1999 Wiley‐Liss, Inc.     

Optical performance of the penguin eye in air and water

Summary Refractive states of three species of penguins (Rockhopper, Gentoo and King) were measured in air and water. Little or no refractive error, with a trend toward slight myopia (less than two dioptres), was found in air in each case. Moderate hyperopia (8–13 dioptres) exists in water. The refractive findings of this study are similar to those of a preliminary study made with the Blackfoot penguin. The relatively small alteration of refractive state associated with the change from air to water (in contrast to an approximate change of 40 dioptres for the human eye) is attributed to the flattened shape of the cornea. The chromatic aberration measured in these species is insufficient to account for the hyperopia found underwater. The maximum reduction of hyperopia resulting from a monochromatic (blue, blue-green) aquatic habitat would only amount to two dioptres. It is speculated that the remaining hyperopia is nullified by an accommodative mechanism.This research was supported by a grant from the National Research Council of Canada. The authors are grateful for the cooperation of the Scottish National Zoological Park, Edinburgh. In particular, the authors would like to thank Mr. Kennedy for assistance in handling the penguins. The authors would like to acknowledge the original observation by Barbara Sivak concerning the flattened appearance of the penguin cornea.     

The eye of the magellanic penguin (Spheniscus magellanicus): structure of the anterior segment

We undertook a light and scanning electron microscopic study of the eye in the Magellanic penguin (Spheniscus magellanicus). The anatomical peculiarities of the eyeball shape in Sphenisciformes have been previously described by others; here, we show that they are accompanied by several modifications in the organization of the anterior segment of the eye. The main change was found in the portion of opaque sclera extending from the cornea to the anterior border of the scleral ossicles, which was much broader than in other avian eyes. This scleral region was made of a very dense fibrous tissue and was as difficult to cut as the ossicles. The corneo-scleral boundary was also different from that of other birds, since the aqueous humor channel and the pectinate ligament were located 1.0-1.5 mm posterior to the cornea. The osseous ring was formed by 13 bones, including three pairs of over- and underplates. There was a single ciliary muscle, with meridionally oriented striated fibers. They were inserted on a circumference along the boundary between the fibrous sclera and the ossicles, far away from the wall of the aqueous humor channel. On their posterior end, the muscle fibers formed a tendinous structure attached to the inner surface of the sclera and to the outer surface of the ciliary body. Only short zonular fibrils were observed. These anatomical features are probably relevant for the adaptation of penguin eyes to vision on land and in the aquatic environment.     

Growth of Jackass penguin chicks (Spheniscus demersus) hand reared on different diets

Jackass penguin chicks from the age of about 10 days were hand reared on different diets: mullet Liza richardsoni , anchovy Engraulis capensis and squid Loligo reynaudi , until they fledged. The weight of food fed and the daily weight increment of the chicks was measured. Excreta were collected every 5–6 days and analysed to establish metabolized energy and metabolic efficiency. Culmen measurements were taken regularly and plotted against age. The age, weight and sum of food fed up to fledging was compared between diets as well as with field data. Compensatory growth was exhibited when stunted chicks on a relatively poor diet had their diet changed to a relatively better diet.     

Arterio-venous heat exchange systems in the Jackass penguin Spheniscus demersus

Extensive arterio-venous associations occur in the head, axillae and legs of the Jackass penguin Spheniscus demersus. These vascular arrangements appear to facilitate counter-current heat exchange. The major heat exchange system in the head is the post orbital rete mirabile formed by the superior orbital artery. Blood from this rete supplies the eye, nasal passages and superficial jaw muscles. Other blood vessels supplying the superficial areas of the head and mouth associate closely with their corresponding veins.
In the axilla the brachial artery divides to form a humeral plexus of parallel running arteries each associating with up to three interlinking veins. Only the marginal vein does not associate with an artery. It appears that a shunt mechanism, which bypasses the veins in the humeral plexus, functions to permit heat loss when required; for instance in a heat-stressed bird breeding or moulting on land.
In both the upper and lower leg all the major arteries and their branches associate closely with corresponding veins.
The development of these arterio-venous associations indicates that Spheniscus demersus is adapted to a cool aquatic environment in which heat retention is of prime importance.     

Optimal conditions for breeding of captive humboldt penguins (Spheniscus humboldti): A survey of British zoos

We surveyed 16 British zoos and bird gardens to assess the optimal conditions for breeding of captive Humboldt penguins (Spheniscus humboldti). We obtained information on population, enclosure, and husbandry characteristics and related these variables to three measures of per capita breeding success, namely, per capita egg productivity, chick productivity, and hatching success (measured as the proportion of eggs laid that hatched). All three fitness measures increased with an increasing number of breeding pairs and total population size but were not related to population density. Once the effect of number of breeding pairs was removed statistically, chick productivity was found to be highest when nesting boxes were lined with sand and gravel instead of alternative substrata such as twigs or vegetation. Hatching success increased with increasing pool size and was highest in enclosures with concrete floors. Adult mortality in zoos was generally low and appeared related to the use of chlorine in freshwater pools and to the presence of other penguin species in Humboldt penguin displays. Several enclosure and husbandry parameters were not variable enough to assess their impact on reproduction of captive Humboldt penguins. Recommendations for optimising conditions for captive breeding of Humboldt penguins include keeping as large a population as possible in a concrete enclosure with a large pool area, while providing sand and gravel as nesting material. Bird density may be important but we did not detect detrimental effects on breeding for densities up to 0.25 birds m^–2. Adult mortality can be minimised by exhibiting Humboldt penguins in single‐species display and avoiding chlorination of pool water. An experimental approach is recommended to confirm the results of this correlational study. Zoo Biol 20:545–555, 2001. © 2002 Wiley‐Liss, Inc.