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.

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

Reduced carnitine level causes death from hypoglycemia: possible involvement of suppression of hypothalamic orexin expression during weaning period.

The mechanism of onset of hypoglycemia in patients with carnitine deficiency has yet to be determined. Using mice with systemic carnitine deficiency (JVS mice), we examined this mechanism, focusing on the weaning period (days 14-28 postpartum). For normal mice, the survival rate was 100%, and no hypoglycemia was observed at all. Gastric lactose began to decrease on day 17, and cellulose increased sharply in amount thereafter. For JVS mice, the survival rate was 77% on day 14 and 28% on day 28. From day 21 on, hypoglycemia was noted. Gastric lactose had disappeared almost completely by day 17, and cellulose was almost undetectable from days 14 to 28. Expression of orexin mRNA in the hypothalamus did not differ between JVS and normal mice on day 14, but was suppressed in JVS mice on days 21 and 28. When JVS mice were fed a carnitine-rich diet, suppression of expression of orexin mRNA in hypothalamus was eliminated, and on day 28 lactose and cellulose were detected in the stomach without hypoglycemia. In conclusion, the suppression of the expression of orexin in the hypothalamus during the weaning period may be involved in the marked anorexia in JVS mice, which eventually leads to death from hypoglycemia.

The rise and fall of the CR1 subfamily in the lineage leading to penguins.

The evolution of penguins has been investigated extensively, although inconclusively, by morphologists, biogeographers and molecular phylogeneticists. We investigated this issue using retroposon analysis of insertions of CR1, which is a member of the LINE (long interspersed element) family, in the genomes of penguins and penguin relatives. The retroposon method is a powerful tool for identifying monophyletic groups. Because retroposons often show different relative frequencies of retroposition during evolution, it is first necessary to identify a certain subgroup that was specifically active during the period when the species in question diverged. Hence, we systematically analyzed many CR1 members isolated from penguin and penguin-related genomes. These CR1s are divided into at least three distinct subgroups that share diagnostic nucleotide insertions and/or deletions, namely, penguin CR1 Sph I, Sph II type A and Sph II type B. The analysis of the inserted retroposons by PCR revealed that different CR1 subfamilies or types had amplified at different rates among different periods during penguin evolution. Namely, the penguin CR1 Sph I subfamily had higher rates of retroposition in a common ancestor of all orders examined in this study or at least in a common ancestor of all extant penguins, and the subfamily Sph II type A also had the same tendency. Therefore, these CR1 members can be used to elucidate the phylogenetic relationships of Sphenisciformes (penguins) among different avian orders. In contrast, the penguin CR1 Sph II type B subfamily had higher rates of retroposition just before and after the emergence of the extant genera in Spheniscidae, suggesting that they are useful for elucidating the intra-relationships among extant penguins. This is the first report for the characterization among the members of CR1 family in avian genomes excluding those of chickens. Hence, this work will be a cornerstone for elucidating the phylogenetic relationships in penguin evolution using the retroposon method.

New candidate species most closely related to penguins.

The phylogenetic position of the order Spenisciformes in Aves remains unclear despite several independent analyses based on morphological and molecular data. To address this issue, we determined the complete mtDNA sequence of rockhopper penguins. The mitochondrial genome, excluding the region from the D-loop to 12SrRNA, was also sequenced for petrel, albatross, frigatebird, loon and grebe, which previous studies suggest are related to penguins. A maximum likelihood analysis of the phylogenetic placement of penguins with 23 birds, including 17 species whose mtDNA sequences were previously reported, suggested that storks are the closest extant relatives of penguins, with 78% and 56% bootstrap supports, depending on the choice of outgroup species. Thus, ciconiiform birds constitute new candidates as the closest extant relatives of penguins (previously proposed candidates were either gaviiform, podicipediform, or procellariiform birds). In addition to this new evidence, our analysis gave evidence to some of ambiguous relationships in the avian tree: our analysis supported a basal split between passerines and other neoavians within Neoaves, and rejected the monophyly of Falconiformes as well as that of loons and grebes.

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.