Gene expression reveals immune response strategies of naïve Hawaiian honeycreepers experimentally infected with introduced avian malaria.

The unprecedented rise in the number of new and emerging infectious diseases in the last quarter century poses direct threats to human and wildlife health. The introduction to the Hawaiian archipelago of Plasmodium relictum and the mosquito vector that transmits the parasite has led to dramatic losses in endemic Hawaiian forest bird species. Understanding how mechanisms of disease immunity to avian malaria may evolve is critical as climate change facilitates increased disease transmission to high elevation habitats where malaria transmission has historically been low and the majority of the remaining extant Hawaiian forest bird species now reside. Here, we compare the transcriptomic profiles of highly susceptible Hawai'i 'amakihi (Chlorodrepanis virens) experimentally infected with P. relictum to those of uninfected control birds from a naïve high elevation population. We examined changes in gene expression profiles at different stages of infection to provide an in-depth characterization of the molecular pathways contributing to survival or mortality in these birds. We show that the timing and magnitude of the innate and adaptive immune response differed substantially between individuals that survived and those that succumbed to infection, and likely contributed to the observed variation in survival. These results lay the foundation for developing gene-based conservation strategies for Hawaiian honeycreepers by identifying candidate genes and cellular pathways involved in the pathogen response that correlate with a bird's ability to recover from malaria infection.

Parallel evolution of gene classes, but not genes: Evidence from Hawai'ian honeycreeper populations exposed to avian malaria.

Adaptation in nature is ubiquitous, yet characterizing its genomic basis is difficult because population demographics cause correlations with nonadaptive loci. Introduction events provide opportunities to observe adaptation over known spatial and temporal scales, facilitating the identification of genes involved in adaptation. The pathogen causing avian malaria, Plasmodium relictum, was introduced to Hawai'i in the 1930s and elicited extinctions and precipitous population declines in native honeycreepers. After a sharp initial population decline, the Hawai'i 'amakihi (Chlorodrepanis virens) has evolved tolerance to the parasite at low elevations where P. relictum exists, and can sustain infection without major fitness consequences. High-elevation, unexposed populations of 'amakihi display little to no tolerance. To explore the genomic basis of adaptation to P. relictum in low-elevation 'amakihi, we genotyped 125 'amakihi from the island of Hawai'i via hybridization capture to 40,000 oligonucleotide baits containing SNPs and used the reference 'amakihi genome to identify genes potentially under selection from malaria. We tested for outlier loci between low- and high-elevation population pairs and identified loci with signatures of selection within low-elevation populations. In some cases, genes commonly involved in the immune response (e.g., major histocompatibility complex) were associated with malaria presence in the population. We also detected several novel candidate loci that may be implicated in surviving malaria infection (e.g., beta-defensin, glycoproteins and interleukin-related genes). Our results suggest that rapid adaptation to pathogens may occur through changes in different immune genes, but in the same classes of genes, across populations.

Transcriptome assembly and differential gene expression of the invasive avian malaria parasite Plasmodium relictum in Hawai'i.

The malaria parasite Plasmodium relictum (lineage GRW4) was introduced less than a century ago to the native avifauna of Hawai'i, where it has since caused major declines of endemic bird populations. One of the native bird species that is frequently infected with GRW4 is the Hawai'i 'amakihi (Chlorodrepanis virens). To achieve a better understanding of the transcriptional activities of this virulent parasite, we performed a controlled challenge experiment of 15 'amakihi that were infected with GRW4. Blood samples containing malaria parasites were collected at two time points (intermediate and peak infection stages) from host individuals that were either experimentally infected by mosquitoes or inoculated with infected blood. We then used RNA sequencing to assemble a high-quality blood transcriptome of P. relictum GRW4, allowing us to quantify parasite expression levels inside individual birds. We found few significant differences (one to two transcripts) in GRW4 expression levels between host infection stages and between inoculation methods. However, 36 transcripts showed differential expression levels among all host individuals, indicating a potential presence of host-specific gene regulation across hosts. To reduce the extinction risk of the remaining native bird species in Hawai'i, genetic resources of the local Plasmodium lineage are needed to enable further molecular characterization of this parasite. Our newly built Hawaiian GRW4 transcriptome assembly, together with analyses of the parasite's transcriptional activities inside the blood of Hawai'i 'amakihi, can provide us with important knowledge on how to combat this deadly avian disease in the future.

Promising protocols for parasites: Metatranscriptomics improves detection of hyperdiverse but low abundance communities.

Genomic technologies continue to shed light on important ecological and evolutionary questions. Nonetheless, these new tools are applied disproportionately in a small fraction of global biodiversity, partly because of technical challenges to studying highly diverse taxa that occur in low abundances in an environment (e.g., marine and microbial communities). As a result, our understanding of ecological and evolutionary processes lags in many taxa. In a From the Cover manuscript in this issue of Molecular Ecology Resources, Galen, Borner, Williamson, Witt, and Perkins (2020) present a novel approach for characterizing diversity that combines metatranscriptomics with rigorous bioinformatic processing to dramatically improve detection and identification of diverse, low-abundance avian blood parasites. Their approach is an exciting application of available tools that increases our potential for a deeper understanding of diversity in other communities of low-abundance, highly diverse taxa.

First Genome Sequence of the Gunnison's Prairie Dog (Cynomys gunnisoni), a Keystone Species and Player in the Transmission of Sylvatic Plague.

Prairie dogs (genus Cynomys) are a charismatic symbol of the American West. Their large social aggregations and complex vocalizations have been the subject of scientific and popular interest for decades. A large body of literature has documented their role as keystone species of western North America's grasslands: They generate habitat for other vertebrates, increase nutrient availability for plants, and act as a food source for mammalian, squamate, and avian predators. An additional keystone role lies in their extreme susceptibility to sylvatic plague (caused by Yersinia pestis), which results in periodic population extinctions, thereby generating spatiotemporal heterogeneity in both biotic communities and ecological processes. Here, we report the first Cynomys genome for a Gunnison's prairie dog (C. gunnisoni gunnisoni) from Telluride, Colorado (USA). The genome was constructed using a hybrid assembly of PacBio and Illumina reads and assembled with MaSuRCA and PBJelly, which resulted in a scaffold N50 of 824 kb. Total genome size was 2.67 Gb, with 32.46% of the bases occurring in repeat regions. We recovered 94.9% (91% complete) of the single copy orthologs using the mammalian Benchmarking Universal Single-Copy Orthologs database and detected 49,377 gene models (332,141 coding regions). Pairwise Sequentially Markovian Coalescent showed support for long-term stable population size followed by a steady decline beginning near the end of the Pleistocene, as well as a recent population reduction. The genome will aid in studies of mammalian evolution, disease resistance, and the genomic basis of life history traits in ground squirrels.

Does the host matter? Variable influence of host traits on parasitism rates.

Parasitism of mammals is ubiquitous, but the processes driving parasite aggregation on hosts are poorly understood, as each system seems to show unique correlations between parasitism and host traits such as sex, age, size and body mass. Genetic diversity is also posited to influence susceptibility to parasitism, and provides a quantifiable measure of an intrinsic unchanging host property, but this link has not been well established. A lack of consistency in host traits predicting parasite heterogeneity may derive from the contribution of environmental factors to parasite aggregation. To evaluate this question, a large dataset was leveraged to explore the relationship between unchanging, intrinsic host traits (heterozygosity and sex), variable host traits (age, length and body mass), and extrinsic factors (sampling date/year and population) and flea presence/absence, abundance and intensity on two species of social burrowing mammal, the black-tailed prairie dog (Cynomys ludovicianus) and the Gunnison's prairie dog (Cynomys gunnisoni). Prairie dogs experience frequent parasitism by fleas, but the distribution of fleas among individuals is highly skewed. In these systems, intrinsic host traits were nuanced in how they predicted flea aggregation on individual prairie dogs, with sex unimportant to parasitism rates and heterozygosity increasing the probability of infection and influencing the number of fleas in divergent ways. Variable host traits interacted with each other and with environmental or geographic stochasticity to influence flea aggregation. Length and age tended to increase parasitism, whereas the effects of body mass and condition were mediated by date and other host traits to produce both positive and negative effects on parasitism. This finding suggests that the factors affecting ectoparasite infection on individuals are complex, even within species. Importantly, there was no correlation between the number of fleas on an individual in one year and the number of fleas on the same individual the next year, supporting the idea that flea aggregation is not driven by unchanging, intrinsic characteristics of the host. Rather, these findings indicate that host traits influence parasitism in nuanced ways, including interactions with environmental characteristics and stochastic factors.