DOI: 10.5061/dryad.69p8cz9cf
title: Gut microbiome of house sparrows during experimental Plasmodium relictum infection

description: The gut microbiome can contribute to host health through defense against
 pathogens. However, links between the host gut microbiome and resistance
 to infection have been primarily investigated in humans and lab-bred
 rodents, so we do not know to what extent this relationship exists in wild
 animals. To fill this knowledge gap, we used an experimental malaria
 inoculation with Plasmodium relictum in wild-caught house sparrows (Passer
 domesticus, n = 18) to examine interactions of the host gut microbiome and
 resistance to avian malaria infection. Before and after inoculation, we
 collected blood samples to quantify circulating parasitemia and fecal
 samples to evaluate the gut microbiome through amplification and
 sequencing of the V4 region of the bacterial 16S rRNA gene. We found that
 gut microbiome alpha diversity (species richness and evenness) and beta
 diversity (community composition) were not significantly different between
 infected and resistant sparrows prior to or during malaria infection.
 However, we identified several indicator ASVs that occurred at higher
 prevalence and proportional abundance in malaria-resistant sparrows, most
 of which were from the family Lactobacillaceae that is hypothesized to
 confer resistance to malaria through inducing the production of anti-α-Gal
 antibodies. Future studies should experimentally manipulate the
 composition of the avian gut microbiome to causally investigate the
 relationship between specific bacterial taxa in the host gut microbiome
 and resistance to avian malaria infection.
description: Prior to and during experimental <em>Plasmodium
 relictum</em> infection, fecal samples were collected from house
 sparrows (<em>Passer domesticus</em>) to evaluate the
 diversity and composition of the gut microbiome. Fecal DNA was extracted
 and the V4 region of the bacterial 16S rRNA gene was amplified. In tandem,
 we collected blood smears before and after inoculation to quantify
 circulating parasite loads. As part of another project, half of the
 sparrows were exposed to mitotane; these sparrows were not included in the
 present analyis.
description: # Gut microbiome of house sparrows during experimental Plasmodium relictum
 infection
 [https://doi.org/10.5061/dryad.69p8cz9cf](https://doi.org/10.5061/dryad.69p8cz9cf) ## Description of the data and file structure Prior to and during experimental *Plasmodium relictum* infection, fecal samples were collected from house sparrows (*Passer domesticus*) to evaluate the diversity and composition of the gut microbiome. Fecal DNA was extracted and the V4 region of the bacterial 16S rRNA gene was amplified. In tandem, we collected blood smears before and after inoculation to quantify circulating parasite loads. As part of another project, half of the sparrows were exposed to mitotane; these sparrows were not included in the present analyis. ### Files and variables #### File: R\_indicator\_ASV\_-\_rarefied.xlsx **Description:** Datafile for indicator species analysis. ##### Variables * HOSP = house sparrow ID * SAMPLE_TYPE = house sparrow (feces) * MALARIA = infected (2) or resistant (1) * MITOTANE = placebo (1) or mitotane (2); mitotane samples were removed from this investigation * TREATMENT = a combination of malaria+mitotane; 1 is placebo & resistant, 2 is placebo & infected * SEX = male (1) or female (2) * WEEK = week of the experiment; 1 & 2 are pre-inoculation, 3 is one week post-inoculation, 4 is at/approaching peak parasitemia * PREW34 = pools weeks 1 & 2 pre-inoculation but keeps week 3 and 4 separate * PREvCRISIS = weeks 1 & 2 pooled, drop week 3 (coded as 'NA'), keeps week 4 * DATE_FECAL = date the fecal sample was obtained * FECAL_MASS = approximate mass of the fecal sample * QUBIT_DNA = DNA concentration as estimated by the QUBIT * ASV1 = frequency of ASV1 * ASV2 = frequency of ASV2  * ASV etc  #### File: R\_phyloseq\_sampledata.xlsx **Description:** Sample-specific data (treatment, physiology, immunity) for respective DNA samples.  ##### Variables * SAMPLE = unique DNA ID * EXTRACTION = extraction number * LABEL = position in the extraction * PCR = PCR group * HOSP = house sparrow ID * SAMPLE TYPE = house sparrow (feces), food type, or paper towel. NAs exist for food samples because physiological data are irrelevant for these samples. * MALARIA = infected (2) or resistant (1) * MITOTANE = placebo (1) or mitotane (2) * TREATMENT = a combination of malaria+mitotane; 1 is placebo & resistant, 2 is placebo & infected, 3 is mitotane & resistant, 4 is mitotane & infected * SEX = male (1) or female (2) * WEEK = week of the experiment; 1 & 2 are pre-inoculation, 3 is one week post-inoculation, 4 is at/approaching peak parasitemia * PREW34 = pools weeks 1 & 2 pre-inoculation but keeps week 3 and 4 separate * PREvCRISIS = weeks 1 & 2 pooled, drop week 3 (coded as 'NA'), keeps week 4.  * DATE_FECAL = date the fecal sample was obtained * FECAL_MASS = approximate mass of the fecal sample * QUBIT_DNA = DNA concentration as estimated by the QUBIT * IGY_OD = optical density units for immunoglobulin Y ELISA; higher numbers reflect higher IgY concentrations * LYSIS = haemolysis score; higher numbers reflect a better ability to lyse foreign erythrocytes * AGGLUTINATION = haemagglutination score; higher numbers reflect a better ability of natural antibodies to coagulate foreign erythrocytes * PARASITEMIA_closest = circulating parasite load that was closest to fecal sampling * PARASITEMIA_dissect = circulating parasite load on 13 days post-inoculation * LYMPHOCYTE = the number of lymphocytes out of the first 100 leukocytes observed (proportional value) * MONOCYTE = the number of monocytes out of the first 100 leukocytes observed (proportional value) * HETEROPHIL = the number of heterophils out of the first 100 leukocytes observed (proportional value) * EOSINOPHIL = the number of eoinophils out of the first 100 leukocytes observed (proportional value) * DATE_SMEAR = ignore this column - incomplete * Notes = notes from quantification - ignore #### File: ASV\_alpha\_diversity\_rarefied1985.csv **Description:** Alpha diversity estimates from mothur. ##### Variables * SAMPLE: DNA sample ID * nseqs: number of sequences retained in the sample; rarefied to 1985 * coverage: number of ASVs that have been observed more than once * sobs: total number of ASVs observed in the sample * invsimpson: "Inverse Simpson" alpha diversity estimate * invsimpson_lci: lower confidence interval for Inverse Simson alpha diversity estimate * invsimpson_hci: upper confidence interval for Inverse Simson alpha diversity estimate * chao: "Chao" alpha diversity estimate * chao_lci: lower confidence interval for Chao alpha diversity estimate * chao_hci: upper confidence interval for Chao alpha diversity estimate * npshannon: "Non-parametric Shannon" diversity estimate * EXTRACTION = extraction number * LABEL = position in the extraction * PCR = PCR group * HOSP = house sparrow ID * SAMPLE TYPE = house sparrow (feces), food type, or paper towel. NAs exist because physiological data are irrelevant for food samples. * MALARIA = infected (2) or resistant (1) * MITOTANE = placebo (1) or mitotane (2) * TREATMENT = a combination of malaria+mitotane; 1 is placebo & resistant, 2 is placebo & infected, 3 is mitotane & resistant, 4 is mitotane & infected * SEX = male (1) or female (2) * WEEK = week of the experiment; 1 & 2 are pre-inoculation, 3 is one week post-inoculation, 4 is at/approaching peak parasitemia * PREW34 = pools weeks 1 & 2 pre-inoculation but keeps week 3 and 4 separate * PREvCRISIS = weeks 1 & 2 pooled, drop week 3 (coded as 'NA'), keeps week 4 * DATE_FECAL = date the fecal sample was obtained * FECAL_MASS = approximate mass of the fecal sample * QUBIT_DNA = DNA concentration as estimated by the QUBIT * IGY_OD = optical density units for immunoglobulin Y ELISA; higher numbers reflect higher IgY concentrations * LYSIS = haemolysis score; higher numbers reflect a better ability to lyse foreign erythrocytes * AGGLUTINATION = haemagglutination score; higher numbers reflect a better ability of natural antibodies to coagulate foreign erythrocytes * PARASITEMIA_closest = circulating parasite load that was closest to fecal sampling * PARASITEMIA_dissect = circulating parasite load on 13 days post-inoculation * LYMPHOCYTE = the number of lymphocytes out of the first 100 leukocytes observed (proportional value) * MONOCYTE = the number of monocytes out of the first 100 leukocytes observed (proportional value) * HETEROPHIL = the number of heterophils out of the first 100 leukocytes observed (proportional value) * EOSINOPHIL = the number of eoinophils out of the first 100 leukocytes observed (proportional value) * DATE_SMEAR = ignore this column - incomplete * Notes = notes from quantification - ignore #### File: R\_phyloseq\_ASV\_taxonomy.xlsx **Description:** Taxonomic classifications for resulting ASVs ##### Variables * ASV = ASV identification number * Doman = taxonomic classification at the resolution of domain * Class = taxonomic classification at the resolution of class * Order = taxonomic classification at the resolution of order * Family = taxonomic classification at the resolution of family * Genus = taxonomic classification at the resolution of genus #### File: R\_phyloseq\_ASV\_abundance.xlsx **Description:** The abundance of each ASV according to DNA sample. ##### Variables * ASV = ASV identification number * RN001 - RN181 = DNA sample ID; column is populated with the abundance of each ASV for the respective sample * ZymoMockDNA = Positive control bacterial community (not an experimental sample; used to assess sequencing error rate) ## Code/software Statistical analysis and figure creation are completed in R Studio v.4.0.2. The packages required for analysis are noted in the script. The script contains a glossary of contents as a guide for users (line 13). Each section includes assumption testing for the statistical test performed and is structured by the question being tested. The Table in the manuscript containing the results the respective statistical analysis is noted. The code to produce each figure is included with the statistical investigation, and is labelled according to the Figure number in the manuscript. Table and Figure numbers are also listed in the "contents" section (line 13).