The orange-tufted sunbird (Cinnyris osea) is a small passerine bird weighing 6–7 g. It has a long, slender, decurved bill (1.4–2.0 cm in length) with a long tongue, which allows it to feed mainly on floral nectar and arthropods . Like other nectarivorous birds, the sunbirds feed on high carbohydrate foods with low-protein content and have high sugar-absorption efficiencies despite the rapid speed that food moves through their gut .
Nicotiana glauca is native to Argentina and Bolivia and is also found in other parts of South America, Hawaii, Australia and in the east Mediterranean region, including Israel. N. glauca is a small tree with long, tubular yellow flowers that bloom almost throughout the year in warm climates. The nectar of N. glauca is rich in sugar with a mean sugar equivalent concentration of 20% ± 0.3%. The nectar contains the toxic pyridine alkaloids nicotine and anabasine in the concentrations of 0.50 ± 0.12 ppm and 5.0 ± 0.8 ppm, respectively . The pollination of N. glauca depends on pollinating vectors because its stamens are shorter than the stigma . N. glauca has a relatively long corolla and, hence, mainly depends on birds with long bills, such as sunbirds and hummingbirds, for pollination. The sunbirds in Israel are the main pollinators of N. glauca (60% of them are legitimate visitors, where they feed on nectar from the front of the flower) .
All methods were performed in accordance with the relevant guidelines and regulations. Sunbirds were captured in Israel according to the regulations and with the permission of Israel Nature and Parks Authority (permission #2016/41432). All experimental procedures and animal care were approved by the Committee of Animal Experimentation of the University of Haifa (permit #477/16, expiration date September/2020). In total, 16 adult sunbirds were captured between December 2017 and January 2018 with mist nets and held in captivity for about 12 weeks. Each bird was held in a separate cage in a room with a controlled temperature (25 °C) and 12h:12h light:dark conditions. After the experiment ended, the birds were set free.
Sunbirds feeding experiment
After capture, the 16 birds were adapted to the laboratory conditions that included a room temperature of 25 °C, 12h:12h light:dark and fed with artificial food (Sunbird nectar special formula for Nectariniidae; Aves & Avian, Lot nr IS240718; Reg.nr. NL113333, Raalte, Netherlands) and water, for a period of four weeks.
After four weeks of captivity, the birds were randomly divided into two groups by assigning eight birds to each group (that is, each bird was assigned randomly, but the cages stayed in the same spot and were not moved). The control group (eight birds) was fed the artificial nectar mentioned above, without any additional nutrients. The treatment group (eight birds) was fed the same artificial nectar with the addition of nicotine and anabasine (Sigma Aldrich, Rehovot, Israel), in concentrations that naturally occur in N. glauca (0.5 ppm and 5 ppm, respectively) (Fig. 1). The concentrations of the pyridine alkaloids were based on Tadmor-Melamed et al. . Fresh artificial nectar and water were supplied every day to both groups.
Excreta (cloacal fluid and faeces) were collected from each of the 16 birds on day 0 (before adding nicotine and anabasine to their feed) and at the end of weeks 2, 4 and 7 (Fig. 1). The excreta collection procedure was as follows: a new, clean piece of baking paper was spread on the bottom of each cage so that the cloacal fluids/faeces would not get contaminated with the cage surface; once the bird left its excreta it was immediately collected using a sterile tip into a sterile Eppendorf tube.
The excreta samples were used for DNA extraction for microbiome analyses (culture-independent) and for isolating nicotine/anabasine–degrading bacteria, as described below.
Cloacal fluids/faeces (excreta) samples for culture-independent analyses were kept at –20 ºC until DNA was extracted using DNeasy Blood and Tissue isolation kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.
16S rRNA gene library The Genomic DNA was PCR-ampliﬁed using primers targeting the V4 region of the 16S rRNA gene. The primers were: CS1_515F (ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGTAA) and CS2_806R (TACGGTAGCAGAGACTTGGTCTGGACTACHVGG-GTWTCTAAT). Primers were synthesized by Sigma Aldrich (Rehovot, Israel) and contained 5’ common sequence tags . The ampliﬁcation was performed in 25 µl reaction volume using the Emerald Amp MAX HS PCR Master Mix (Takara Bio Inc., Otsu, Shiga, Japan). Primer concentrations were 0.5 ng/µl. The PCR experimental conditions were as follows: 95 °C for 5 min, followed by 28 cycles of 30 s at 95 °C, 45 s at 55 °C, and 30 s at 68 °C. A ﬁnal elongation step of 7 min at 68 °C was included. The ampliﬁcation products were veriﬁed by running in agarose gel electrophoresis. The PCR products were stored at −20 °C.
Illumina sequencing. Illumina MiniSeq sequencing was performed at the DNA Services Facility, University of Illinois, Chicago. The sequencing protocol was followed exactly as described by Aizenberg et al . Before sequencing the samples, a second PCR amplification was performed in a 10 μl reaction in 96 wells plate. The master mix used for the reaction was made using the 2X AccuPrime SuperMix II (Thermo Fisher Scientific, Massachusetts, United States). A final concentration of 400 nM of each primer was used, and each respective well in the 96 wells plate received a separate primer set with a unique 10-base barcode (Fluidigm, South San Francisco, CA, USA; item #100-4876). The unique barcodes in separate reactions were used for the positive control and a second no-template control reaction with only Access Array Barcode library primers were used. The amplification conditions were 95 °C for 5 min, followed by 8 cycles at 95 °C for 30s, 60 °C for 30s and 68 °C for 30s. A final, 7 min elongation step was performed at 68 °C. The amplified products of positive and negative controls and selected samples were validated using Qubit fluorometric quantitation with the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, California, United States. After finding the quality of amplification, the samples were collected in equal volume and purified in solid phase reversible immobilization (SPRI). The final quality control was performed using Agilent 2200 TapeStation and Qubit analysis, prior to dilution to 6 pM for emulsion PCR. Pooled, diluted libraries were sequenced on an Illumina MiSeq instrument, using the MiSeq 600-cycle sequencing kit version 3, and analyzed with Casava 1.8 (pipeline 1.8). The reads were 150 nucleotides in length and PhiX DNA serves as a spike-in control. Barcode sequences from Fluidigm were provided to the MiSeq server, and sequences were automatically binned according to 10-base multiplex identifier sequences. Raw reads were recovered as FASTQ files.
Sequence analysis. Bioinformatics was performed using the DADA2 pipeline. Data analysis was performed on unrarefied data. Amplicon sequence variants (ASV) were selected using DADA2 . ASVs were filtered when they were not assigned to the Domain Bacteria or were assigned to the family Mitochondria, the class of Chloroplast or the phylum of Cyanobacteria/Chloroplast. Taxonomy was assigned using the Ribosomal Database Project 16S rRNA gene sequence database . All subsequent ecological analyses were completed using PhyloSeq and other R packages .
Sequences were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject accession number PRJNA548382.
Statistical analysis. The sample rarefaction analysis was performed using the iNEXT R package . The non-metric multidimensional scaling analysis (nMDS), based on the Bray-Curtis dissimilarity matrix, was analysed in PRIMER7 in order to test whether the bacterial community differs between the treatment and control groups. An analysis of similarity (ANOSIM) was performed between the treatment and control groups at various time intervals. The Shannon H′ diversity index was analysed in PAST. Mixed model ANOVA was carried out to determine the effects of time and treatment on the Shannon H diversity index of the microbiome using SPSS23. No violations of data sphericity were detected (Mauchly's test of sphericity: p > 0.05). Similarity percentages routine (SIMPER) was applied using the PRIMER7 software. The average Bray-Curtis dissimilarity in the structure of the microbial community between the control and treatment groups was calculated for each time point separately. The contributions from each bacterial genus to the overall dissimilarity between the groups (with a 90% cut-off) are presented in the results.
Isolation of nicotine/anabasine–degrading bacteria. The nicotine/anabasine–degrading bacteria were isolated at two different sampling time points: (i) cloacal excreta were collected from the naïve birds, which were fed with artificial nectar at day 0 (0 weeks control group, 0WC; 0 weeks treatment group, 0WT; n = 8 at each group; hereafter, weeks control and weeks treatment will be mentioned as WC and WT, respectively) (Fig. 1); (ii) four weeks after starting to add nicotine and anabasine to the special food in the treatment group (4WC and 4WT) (Fig. 1). The excreta were collected as described above. Collected samples were immediately cultured. For enriching nicotine- or anabasine-degrading bacteria, we used the M9 minimal medium (Na2HPO4.7H2O – 64g, KH2PO4 – 15g, NaCl – 2.5g, H2O – 1L), sterilized by autoclaving. To 200 ml of this mixture, we added 700 ml water, 2 ml sterile 1M MgSO4 and 100 µl 1M CaCl2; and the whole solution was adjusted to 1 l with H2O, with the addition of either 0.1% nicotine or 0.1% anabasine as the only carbon and nitrogen sources. Samples of 100 µl of the collected faeces were incubated in 500 µl M9 minimal medium at 37 ºC for 3 to 6 hours. After this enrichment incubation, samples were inoculated on agar plates with the same medium but with the addition of 2% agar. Plates were incubated at 37 ºC for 5 to 7 days.
Nicotine- and anabasine-degrading bacterial colonies were picked and streaked five times on Luria-Bertani (LB, HiMedia Laboratories, Mumbai, India) agar plates. Their ability to grow on nicotine or anabasine as the only carbon and nitrogen sources was verified again by growing them on M9 agar plates with nicotine or anabasine as carbon or nitrogen sources. Pure bacterial isolates were kept in LB broth with 30% glycerol at –80 ºC.
The bacterial isolates were identified by amplifying and sequencing a 1501-bp internal fragment of 16S rRNA gene, in accordance with Senderovich et al. . Purified PCR products were sequenced at MCLAB (South San Francisco, CA, USA) and analyses of all sequences was carried out using the EzTaxon website (http://eztaxon-e.ezbiocloud.net/) . The sequences were deposited in the GenBank database under the accession numbers: MK348690–MK348835.