The Hawaiian and Mediterranean Monk Seal populations have suffered dramatic declines in numbers in recent years. Population surveys of Hawaiian Monk Seals indicate a decrease of 50% since the 1950's with approximately 1450 animals remaining. Regardless of the cause, the population decline itself is of great concern since the ability of a species to withstand extinction can be linked to the amount of genetic variation possessed by that species. The maintenance of genetic diversity in a species is one of the central concepts in conservation biology because it is believed to contribute to both short and long-term survivability. It is hypothesized that a highly variable gene pool may act as a reservoir for adaptation during long-term environmental changes and as a defense against short-term potentially catastrophic events such as the introduction of a new pathogen. Previous studies examining the genetic diversity of monk seal populations have focused on population marker genes rather than those involved in adaptation to ecological insults. While useful in examining population ancestry, these studies are unable to examine the complex influence of infectious disease on population dynamics and host susceptibility. This can only be achieved by examining genes encoding proteins that are actively involved in host-pathogen interactions.
The major histocompatibility complex (MHC) is a family of highly polymorphic genes encoding a set of transmembrane proteins that are critical to the generation of immune responses. These cell surface glycoproteins play a key role in the initiation of immune response by binding foreign peptides and presenting them to T-cells. The polymorphism of these MHC-encoded proteins ultimately determines the repertoire of antigenic determinants to which an individual is capable of responding. The high level of MHC genetic variation found in most mammals is thought to be an adaptation to the large number of pathogens encountered by natural populations. It has also been suggested that infectious disease epidemics of the past have played a central role in determining the MHC allele and haplotype frequencies observed in populations today. This study was performed to help develop techniques by which we could compare and contrast the MHC genotypes of various monk seal populations.
The first step in this process was to characterize monk seal class I MHC genes so that species-specific reagents could be designed for genotyping larger numbers of animals. To facilitate the amplification of full-length class I MHC gene transcripts a cDNA population was constructed using RACE (rapid amplification of cDNA ends) technology. The template for these constructs was total cellular RNA isolated from the peripheral blood mononuclear cells (PBMCs) of two monk seals in rehabilitation. Degenerate class I-specific primers were designed based on nucleotide alignments of equine, bovine, porcine, canine, and human RNA-derived MHC class I gene sequences. These primers were used to amplify near full-length gene sequences from the RACE cDNA populations described above. Bands representing PCR products of the predicted size were excised from the gel, extracted, and ligated into a T/A-type cloning vector. The nucleotide sequences of the cloned monk seal class I MHC products were determined by dideoxy nucleotide methodology using an automated sequencer, and were compared using sequence alignment software programs.
These techniques were successful in amplifying monk seal-specific MHC class I gene sequences in both animals. Results from the sequence analyses indicated that, in contrast to other pinnipeds, only a small number of MHC class I gene transcripts could be identified in each individual monk seal. Furthermore, the amount of class I transcript sequence variation both within an individual, and between individuals, was extremely low. To confirm this finding, a technique called denaturing gradient gel electrophoresis (DGGE) was used to compare the degree of class I nucleotide variation between individuals. Specific primer pairs were used in PCR to amplify sequences representing the two exons encoding the putative MHC class I peptide-binding site. The MHC class I genotype of both exons was compared across 6 monk seals in rehabilitation. The results confirmed the initial findings, identifying only 3 different exon 2 sequences, and two different exon 3 sequences in each individual. The gels also allowed comparison of MHC class I genotype between animals, and clearly showed that class I variability between individuals was extremely limited.
In combination, these findings raise concern regarding the amount of class I MHC diversity in the monk seal population. These results should however, be interpreted with caution because of the low number of individuals included in the study. It is possible that these animals originated from a single breeding group, and thus the lack of MHC heterogeneity between individuals may not be representative of the greater monk seal population. A broader class I MHC genotype study of monk seals is required before conclusions can be made regarding immunogenetic diversity of the broader population. The molecular characterization of the monk seal MHC reported here, and its application in adapting a rapid MHC genotyping technique, is however extremely useful. The variability and immunological importance of the genes in the MHC complex makes them ideal candidates for identifying parental lineage, evaluating genetic diversity and predicting susceptibility to specific pathogens. The results of a broader study will allow us to estimate whether a lack of immune system diversity may, either now or in the future, contribute to an increased susceptibility to infectious disease in this fragile population. In addition, this information will be essential for management decisions regarding animal relocations and prophylactic strategies against infectious disease in this uniquely challenged population of pinnipeds.
The authors would like to thank the Morris Animal Foundation for providing funds to support this study.