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Hawaiian honeycreepers (Drepanidinae) have evolved in the absence of mosquitoes for over five million years. Through human activity, mosquitoes were introduced to the Hawaiian archipelago less than 200 years ago. Mosquito-vectored diseases such as avian malaria caused by Plasmodium relictum and Avipoxviruses have greatly impacted these vulnerable species. Susceptibility to these diseases is variable among and within species. Due to their function in adaptive immunity, the role of major histocompatibility complex genes (Mhc) in disease susceptibility is under investigation. In this study, we evaluate gene organization and levels of diversity of Mhc class II β chain genes (exon 2) in a captive-reared family of Hawaii 'amakihi (Hemignathus virens). A total of 233 sequences (173 bp) were obtained by PCR+1 amplification and cloning, and 5720 sequences were generated by Roche 454 pyrosequencing. We report a total of 17 alleles originating from a minimum of 14 distinct loci. We detected three linkage groups that appear to represent three distinct haplotypes. Phylogenetic analysis revealed one variable cluster resembling classical Mhc sequences (DAB) and one highly conserved, low variability cluster resembling non-classical Mhc sequences (DBB). High net evolutionary divergence values between DAB and DBB resemble that seen between chicken BLB system and YLB system genes. High amino acid identity among non-classical alleles from 12 species of passerines (DBB) and four species of Galliformes (YLB) was found, suggesting that these non-classical passerine sequences may be related to the Galliforme YLB sequences.

The objective of this study was to estimate the number and degree of variation of Mhc class II β chain genes and alleles in a family of Hawaiian honeycreepers (`Amakihi, Hemignathus virens). The parents were bred in captivity and produced eight progeny. Partial antigen binding region (173 bp) fragments were produced via PCR+1 cloning and sequenced on a Beckman CEQ system, and two Roche 454 platform pyrosequencing runs. A total of 302 sequences were obtained via cloning. After quality filtering and removal of chimeras, a total of 2762 sequences were retained from 454 run 1 and 3958 sequences from 454 run 2. Unique sequences detected

While most species of Hawaiian honeycreeper are highly susceptible to avian malaria, populations of the Hawaii amakihi (Hemignathus virens) are thriving at low elevations despite high infection rates of Plasmodium relictum. Due to their critical function in adaptive immunity, proteins encoded by Mhc genes may play a role in the recently evolved host defense mechanisms in these populations. A 454 Roche platform was used to generate 44,014 sequences representing 158 Mhc alleles from 46 captive amakihi involved in experimental challenges to avian malaria and 88 wild amakihi originating from Hawaii Island. Analysis of an NJ tree revealed 6 clusters of alleles occurring most often in surviving amakihi, and a significant association between survivorship and the presence of one or more alleles from these clusters was detected (p = 0.00006). Several of these alleles were also found in higher frequencies in low vs. high elevation populations. A similar analysis of 6,417 sequences representing 31 Mhc alleles from 19 experimentally infected i`iwi (Vestiaria coccinea) revealed a significant association between survivorship and the presence of one or more alleles from two clusters (p = 0.02). This is the first documentation of an Mhc association with survival to malaria in Hawaiian honeycreepers and is expected to enhance our understanding of host-parasite interactions in this natural disease system while also providing a predictive tool for conservation and management.

A better understanding of host defenses against malaria is important, but most studies have traditionally focused on resistance and/or immunological mechanisms that limit parasite burden. Host tolerance mechanisms are thought to minimize physiological damage caused by both the parasite itself and immunopathology without directly affecting parasite burden. On Hawai`i Island, low elevation populations of amakihi (Hemignathus virens) appear to have recently evolved tolerance mechanisms against avian malaria (Plasmodium relictum). Under comparable parasite loads, low elevation amakihi have lower mortality, greater fitness, lower reticulocyte levels and lower heterophil to lymphocyte ratios than high elevation amakihi. Thus, distinct host defense mechanisms are likely operating in low vs. high elevation amakihi. To search for genes involved in host defense, AFLP analysis was completed. Group significance tests (Bionumerics v 2.0) reveal genetic distinction between groups (p = 0.000). Band frequency comparisons between low and high elevation `amakihi (Fisher’s exact test and Pearson’s chi-square test with Yate’s continuity correction, R v 2.11.1) revealed ten band classes with significantly higher (p < 0.05) frequencies in low vs. high elevation birds and 13 band classes with higher frequencies in high vs. low elevation birds. Isolation and sequencing of these candidate bands is underway and may provide a better understanding of host defense mechanisms in natural populations.