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




Summary: Of the estimated 55 Hawaiian honeycreepers (subfamily Carduelinae) only 17 species remain, 9 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 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 some 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 qPCR. 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.




Required software:
Qiime2 version 2020.11




R version 4.2.1




Code:
Qiime2_Pipeline.txt - Code for processing raw sequencing reads using Qiime2




CleanCode.R - R script for post-dada2 processing, analysis, and data visualization




Data:
edited-tax-Run1.tsv - Qiime2 generated ASV taxonomy table, separating ASVs by kingdom, phylum, class, order, family, genus, and species; used in generating phyloseq object for statistics and figures




out_table.tsv - Qiime2 generated counts of each ASV for each microbiome sample; used in generating phyloseq object for statistics and figures




tree.nwk - Qiime2 generated phylogenetic tree of representative ASV sequences; used in generating phyloseq object for statistics and figures




DeSeq2_normtab.tsv - DeSeq2 generated ASV abundance counts normalized to each sample’s library size; used in statistical analysis 




Prevalence.tsv - Avian malaria prevalence at the site where the sample was collected; used in statistical analysis




Metadata.tsv - Metadata for all sequenced samples (variables in table below); used in generating phyloseq object for statistics and figures
Column Name - Description
        SampleID - Unique identifier for each set of samples
        Host - Bird species sampled
        Age - Whether bird sampled was an adult or juvenile 
        Sex - Sex of the bird sampled 
        Region - Geographic region of sample collection site
        SiteCode - Sample collection site
        Date - Sample collection date
        Elevation - Sampling site elevation (meters)
        qPCR_Run1 - Ct values for first qPCR run used to detect infection
        qPCR_Run2 - Ct values for second qPCR run used to detect infection
        AvgCt - Averaged qPCR Ct value
        RelInf - Relative parasitemia intensity calculated using 1/(2(avgCt-25))
        Status - Detailed malaria infection status (uninfected, chronic=chronically infected, active=actively infected)
        PosOrNeg - Malaria infection status
        SampleorControl - Indicates sample vs negative controls (S=sample, C=negative control)        


Other resources: 
Raw sequence reads are available from NCBI SRA under Project number PRJNA794822