Linking avian malaria parasitemia estimates from quantitative PCR and microscopy reveals new infection patterns in Hawai'i.

Plasmodium parasites infect thousands of species and provide an exceptional system for studying host-pathogen dynamics, especially for multi-host pathogens. However, understanding these interactions requires an accurate assay of infection. Assessing Plasmodium infections using microscopy on blood smears often misses infections with low parasitemias (the fractions of cells infected), and biases in malaria prevalence estimates will differ among hosts that differ in mean parasitemias. We examined Plasmodium relictum infection and parasitemia using both microscopy of blood smears and quantitative polymerase chain reaction (qPCR) on 299 samples from multiple bird species in Hawai'i and fit models to predict parasitemias from qPCR cycle threshold (Ct) values. We used these models to quantify the extent to which microscopy underestimated infection prevalence and to more accurately estimate infection patterns for each species for a large historical study done by microscopy. We found that most qPCR-positive wild-caught birds in Hawaii had low parasitemias (Ct scores ≥35), which were rarely detected by microscopy. The fraction of infections missed by microscopy differed substantially among eight species due to differences in species' parasitemia levels. Infection prevalence was likely 4-5-fold higher than previous microscopy estimates for three introduced species, including Zosterops japonicus, Hawaii's most abundant forest bird, which had low average parasitemias. In contrast, prevalence was likely only 1.5-2.3-fold higher than previous estimates for Himatione sanguinea and Chlorodrepanis virens, two native species with high average parasitemias. Our results indicate that relative patterns of infection among species differ substantially from those observed in previous microscopy studies, and that differences depend on variation in parasitemias among species. Although microscopy of blood smears is useful for estimating the frequency of different Plasmodium stages and host attributes, more sensitive quantitative methods, including qPCR, are needed to accurately estimate and compare infection prevalence among host species.

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.

Microbiomes associated with avian malaria survival differ between susceptible Hawaiian honeycreepers and sympatric malaria-resistant introduced birds.

Of the estimated 55 Hawaiian honeycreepers (subfamily Carduelinae) only 17 species remain, nine of which the International Union for Conservation of Nature considers endangered. Among the most pressing threats to honeycreeper survival is avian malaria, caused by the introduced blood parasite Plasmodium relictum, which is increasing in distribution in Hawai'i as a result of climate change. Preventing further honeycreeper decline will require innovative conservation strategies that confront malaria from multiple angles. Research on mammals has revealed strong connections between gut microbiome composition and malaria susceptibility, illuminating a potential novel approach to malaria control through the manipulation of gut microbiota. One honeycreeper species, Hawai'i 'amakihi (Chlorodrepanis virens), persists in areas of high malaria prevalence, indicating they have acquired some level of immunity. To investigate if avian host-specific microbes may be associated with malaria survival, we characterized cloacal microbiomes and malaria infection for 174 'amakihi and 172 malaria-resistant warbling white-eyes (Zosterops japonicus) from Hawai'i Island using 16S rRNA gene metabarcoding and quantitative polymerase chain reaction. Neither microbial alpha nor beta diversity covaried with infection, but 149 microbes showed positive associations with malaria survivors. Among these were Escherichia and Lactobacillus spp., which appear to mitigate malaria severity in mammalian hosts, revealing promising candidates for future probiotic research for augmenting malaria immunity in sensitive endangered species.

Optimizing trilateration estimates for tracking fine-scale movement of wildlife using automated radio telemetry networks.

A major advancement in the use of radio telemetry has been the development of automated radio tracking systems (ARTS), which allow animal movements to be tracked continuously. A new ARTS approach is the use of a network of simple radio receivers (nodes) that collect radio signal strength (RSS) values from animal-borne radio transmitters. However, the use of RSS-based localization methods in wildlife tracking research is new, and analytical approaches critical for determining high-quality location data have lagged behind technological developments. We present an analytical approach to optimize RSS-based localization estimates for a node network designed to track fine-scale animal movements in a localized area. Specifically, we test the application of analytical filters (signal strength, distance among nodes) to data from real and simulated node networks that differ in the density and configuration of nodes. We evaluate how different filters and network configurations (density and regularity of node spacing) may influence the accuracy of RSS-based localization estimates. Overall, the use of signal strength and distance-based filters resulted in a 3- to 9-fold increase in median accuracy of location estimates over unfiltered estimates, with the most stringent filters providing location estimates with a median accuracy ranging from 28 to 73 m depending on the configuration and spacing of the node network. We found that distance filters performed significantly better than RSS filters for networks with evenly spaced nodes, but the advantage diminished when nodes were less uniformly spaced within a network. Our results not only provide analytical approaches to greatly increase the accuracy of RSS-based localization estimates, as well as the computer code to do so, but also provide guidance on how to best configure node networks to maximize the accuracy and capabilities of such systems for wildlife tracking studies.

Individual and seasonal variation in the movement behavior of two tropical nectarivorous birds.

BACKGROUND: Movement of animals directly affects individual fitness, yet fine spatial and temporal resolution movement behavior has been studied in relatively few small species, particularly in the tropics. Nectarivorous Hawaiian honeycreepers are believed to be highly mobile throughout the year, but their fine-scale movement patterns remain unknown. The movement behavior of these crucial pollinators has important implications for forest ecology, and for mortality from avian malaria (Plasmodium relictum), an introduced disease that does not occur in high-elevation forests where Hawaiian honeycreepers primarily breed. METHODS: We used an automated radio telemetry network to track the movement of two Hawaiian honeycreeper species, the 'apapane (Himatione sanguinea) and 'i'iwi (Drepanis coccinea). We collected high temporal and spatial resolution data across the annual cycle. We identified movement strategies using a multivariate analysis of movement metrics and assessed seasonal changes in movement behavior. RESULTS: Both species exhibited multiple movement strategies including sedentary, central place foraging, commuting, and nomadism , and these movement strategies occurred simultaneously across the population. We observed a high degree of intraspecific variability at the individual and population level. The timing of the movement strategies corresponded well with regional bloom patterns of 'ohi'a (Metrosideros polymorpha) the primary nectar source for the focal species. Birds made long-distance flights, including multi-day forays outside the tracking array, but exhibited a high degree of fidelity to a core use area, even in the non-breeding period. Both species visited elevations where avian malaria can occur but exhibited little seasonal change in elevation (< 150 m) and regularly returned to high-elevation roosts at night. CONCLUSIONS: This study demonstrates the power of automated telemetry to study complex and fine-scale movement behaviors in rugged tropical environments. Our work reveals a system in which birds can track shifting resources using a diverse set of movement behaviors and can facultatively respond to environmental change. Importantly, fidelity to high-elevation roosting sites minimizes nocturnal exposure to avian malaria for far-ranging individuals and is thus a beneficial behavior that may be under high selection pressure.

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.

Stable isotope analysis of multiple tissues from Hawaiian honeycreepers indicates elevational movement.

We have limited knowledge of the patterns, causes, and prevalence of elevational migration despite observations of seasonal movements of animals along elevational gradients in montane systems worldwide. While a third of extant Hawaiian landbird species are estimated to be elevational migrants this assumption is based primarily on early naturalist's observations with limited empirical evidence. In this study, we compared stable hydrogen isotopes (δ2H) of metabolically inert (feathers) and active (blood plasma, red blood cells) tissues collected from the same individual to determine if present day populations of Hawaiian honeycreepers undergo elevational movements to track areas of seasonally high flower bloom that constitute significant food resources. We also measured stable carbon isotopes (δ13C) and stable nitrogen isotopes (δ15N) to examine potential changes in diet between time periods. We found that the majority of 'apapane (Himatione sanguinea) and Hawai'i 'amakihi (Chlorodrepanis virens) captured at high elevation, high bloom flowering sites in the fall were not year-round residents at the capture locations, but had molted their feathers at lower elevations presumably in the summer after breeding. δ2H values of feathers for all individuals sampled were higher than blood plasma isotope values after accounting for differences in tissue-specific discrimination. We did not find a difference in the propensity of elevational movement between 'apapane and Hawai'i 'amakihi, even though the 'amakihi is considered more sedentary. However, consistent with a more generalist diet, δ15N values indicated that Hawai'i 'amakihi had a more diverse diet across trophic levels than 'apapane, and a greater reliance on nectar in the fall. We demonstrate that collecting multiple tissue samples, which grow at different rates or time periods, from a single individual can provide insights into elevational movements of Hawaiian honeycreepers over an extended time period.

Loss of cultural song diversity and the convergence of songs in a declining Hawaiian forest bird community.

The effects of population decline on culturally transmitted behaviours in animals have rarely been described, but may have major implications to population viability. Learned vocal signals in birds are of critical importance to behaviours associated with reproduction, intrasexual interactions and group cohesion, and the complexity of vocal signals such as song can serve as an honest signal of an individual's quality as well as the viability of a population. In this study, we examined how rapid population declines recently experienced by Hawaiian honeycreepers on the island of Kaua'i (USA) may have influenced the diversity, complexity and similarity of learned honeycreeper songs. We analysed the acoustic characteristics of songs recorded during three time periods over a 40-year time frame for three species of declining Kaua'i honeycreepers. We detected a loss of song complexity and diversity over the 40-year time period that paralleled dramatic population declines. Concurrent with the loss of complexity, we also found that the acoustic characteristics of the three honeycreepers' songs became more similar to one another. To our knowledge, this is the first documentation of convergence of acoustic characteristics among rapidly declining species. The reduction in song complexity and diversity and convergence of songs not only signals a loss of culturally transmitted behaviours in these endemic Hawaiian honeycreepers, but also potential challenges to the recovery of these rapidly declining species. Moreover, the present study highlights that there is a 'hidden' cost to declining populations beyond just the loss of individuals that is not often considered, the loss of culturally transmitted social behaviours.

The effect of isolation, fragmentation, and population bottlenecks on song structure of a Hawaiian honeycreeper.

Little is known about how important social behaviors such as song vary within and among populations for any of the endemic Hawaiian honeycreepers. Habitat loss and non-native diseases (e.g., avian malaria) have resulted in isolation and fragmentation of Hawaiian honeycreepers within primarily high elevation forests. In this study, we examined how isolation of Hawai'i 'amakihi (Chlorodrepanis virens) populations within a fragmented landscape influences acoustic variability in song. In the last decade, small, isolated populations of disease tolerant 'amakihi have been found within low elevation forests, allowing us to record 'amakihi songs across a large elevational gradient (10-1800 m) that parallels disease susceptibility on Hawai'i island. To understand underlying differences among populations, we examined the role of geographic distance, elevation, and habitat structure on acoustic characteristics of 'amakihi songs. We found that the acoustic characteristics of 'amakihi songs and song-type repertoires varied most strongly across an elevational gradient. Differences in 'amakihi song types were primarily driven by less complex songs (e.g., fewer frequency changes, shorter songs) of individuals recorded at low elevation sites compared to mid and high elevation populations. The reduced complexity of 'amakihi songs at low elevation sites is most likely shaped by the effects of habitat fragmentation and a disease-driven population bottleneck associated with avian malaria, and maintained through isolation, localized song learning and sharing, and cultural drift. These results highlight how a non-native disease through its influence on population demographics may have also indirectly played a role in shaping the acoustic characteristics of a species.

Connecting the dots: Stopover strategies of an intercontinental migratory songbird in the context of the annual cycle.

The phases of the annual cycle for migratory species are inextricably linked. Yet, less than five percent of ecological studies examine seasonal interactions. In this study, we utilized stable hydrogen isotopes to geographically link individual black-and-white warblers (Mniotilta varia) captured during spring migration with breeding destinations to understand a migrant's stopover strategy in the context of other phases of the annual cycle. We found that stopover strategy is not only a function of a bird's current energetic state, but also the distance remaining to breeding destination and a bird's time-schedule, which has previously been linked to habitat conditions experienced in the preceding phase of the annual cycle. Birds in close proximity to their breeding destination accumulate additional energy reserves prior to arrival on the breeding grounds, as reflected by higher migratory condition upon arrival, higher refueling rates measured via blood plasma metabolites, and longer stopover durations compared to birds migrating to breeding destinations farther from the stopover site. However, late birds near their breeding destination were more likely to depart on the day of arrival (i.e., transients), and among birds that stopped over at the site, the average duration of stopover was almost half the time of early conspecifics, suggesting late birds are trying to catch-up with the overall time-schedule of migration for optimal arrival time on the breeding grounds. In contrast, birds with long distances remaining to breeding destinations were more likely to depart on the day of arrival and primarily used stopover to rest before quickly resuming migration, adopting similar strategies regardless of a bird's time-schedule. Our study demonstrates that migrants adjust their en route strategies in relation to their time-schedule and distance remaining to their breeding destination, highlighting that strategies of migration should be examined in the context of other phases of the annual cycle.

Seasonal gene expression in a migratory songbird.

The annual migration of a bird can involve thousands of kilometres of nonstop flight, requiring accurately timed seasonal changes in physiology and behaviour. Understanding the molecular mechanisms controlling this endogenous programme can provide functional and evolutionary insights into the circannual biological clock and the potential of migratory species to adapt to changing environments. Under naturally timed photoperiod conditions, we maintained captive Swainson's thrushes (Catharus ustulatus) and performed RNA sequencing (RNA-Seq) of the ventral hypothalamus and optic chiasma to evaluate transcriptome-wide gene expression changes of individuals in migratory condition. We found that 188 genes were differentially expressed in relation to migratory state, 86% of which have not been previously linked to avian migration. Focal hub genes were identified that are candidate variables responsible for the occurrence of migration (e.g. CRABP1). Numerous genes involved in cell adhesion, proliferation and motility were differentially expressed (including RHOJ, PAK1 and TLN1), suggesting that migration-related changes are regulated by seasonal neural plasticity.

Mapping migration in a songbird using high-resolution genetic markers.

Neotropic migratory birds are declining across the Western Hemisphere, but conservation efforts have been hampered by the inability to assess where migrants are most limited-the breeding grounds, migratory stopover sites or wintering areas. A major challenge has been the lack of an efficient, reliable and broadly applicable method for measuring the strength of migratory connections between populations across the annual cycle. Here, we show how high-resolution genetic markers can be used to identify genetically distinct groups of a migratory bird, the Wilson's warbler (Cardellina pusilla), at fine enough spatial scales to facilitate assessing regional drivers of demographic trends. By screening 1626 samples using 96 highly divergent single nucleotide polymorphisms selected from a large pool of candidates (~450 000), we identify novel region-specific migratory routes and timetables of migration along the Pacific Flyway. Our results illustrate that high-resolution genetic markers are more reliable, precise and amenable to high throughput screening than previously described intrinsic marking techniques, making them broadly applicable to large-scale monitoring and conservation of migratory organisms.

El Niño-Southern Oscillation is linked to decreased energetic condition in long-distance migrants.

Predicting how migratory animals respond to changing climatic conditions requires knowledge of how climatic events affect each phase of the annual cycle and how those effects carry-over to subsequent phases. We utilized a 17-year migration dataset to examine how El Niño-Southern Oscillation climatic events in geographically different regions of the Western hemisphere carry-over to impact the stopover biology of several intercontinental migratory bird species. We found that migratory birds that over-wintered in South America experienced significantly drier environments during El Niño years, as reflected by reduced Normalized Difference Vegetation Index (NDVI) values, and arrived at stopover sites in reduced energetic condition during spring migration. During El Niño years migrants were also more likely to stopover immediately along the northern Gulf coast of the southeastern U.S. after crossing the Gulf of Mexico in small suboptimal forest patches where food resources are lower and migrant density often greater than larger more contiguous forests further inland. In contrast, NDVI values did not differ between El Niño and La Niña years in Caribbean-Central America, and we found no difference in energetic condition or use of coastal habitats for migrants en route from Caribbean-Central America wintering areas. Birds over-wintering in both regions had consistent median arrival dates along the northern Gulf coast, suggesting that there is a strong drive for birds to maintain their time program regardless of their overall condition. We provide strong evidence that not only is the stopover biology of migratory landbirds influenced by events during the previous phase of their life-cycle, but where migratory birds over-winter determines how vulnerable they are to global climatic cycles. Increased frequency and intensity of ENSO events over the coming decades, as predicted by climatic models, may disproportionately influence long-distance migrants over-wintering in South America.

Mortality gradients within and among dominant plant populations as barometers of ecosystem change during extreme drought.

Understanding patterns of plant population mortality during extreme weather events is important to conservation planners because the frequency of such events is expected to increase, creating the need to integrate climatic uncertainty into management. Dominant plants provide habitat and ecosystem structure, so changes in their distribution can be expected to have cascading effects on entire communities. Observing areas that respond quickly to climate fluctuations provides foresight into future ecological changes and will help prioritize conservation efforts. We investigated patterns of mortality in six dominant plant species during a drought in the southwestern United States. We quantified population mortality for each species across its regional distribution and tested hypotheses to identify ecological stress gradients for each species. Our results revealed three major patterns: (1) dominant species from diverse habitat types (i.e., riparian, chaparral, and low- to high-elevation forests) exhibited significant mortality, indicating that the effects of drought were widespread; (2) average mortality differed among dominant species (one-seed juniper[Juniperus monosperma (Engelm.) Sarg.] 3.3%; manzanita[Arctostaphylos pungens Kunth], 14.6%; quaking aspen[Populus tremuloides Michx.], 15.4%; ponderosa pine[Pinus ponderosa P. & C. Lawson], 15.9%; Fremont cottonwood[Populus fremontii S. Wats.], 20.7%; and pinyon pine[Pinus edulis Engelm.], 41.4%); (3) all dominant species showed localized patterns of very high mortality (24-100%) consistent with water stress gradients. Land managers should plan for climatic uncertainty by promoting tree recruitment in rare habitat types, alleviating unnatural levels of competition on dominant plants, and conserving sites across water stress gradients. High-stress sites, such as those we examined, have conservation value as barometers of change and because they may harbor genotypes that are adapted to climatic extremes.