Cavia

The term “aerosol” is used herein to describe respiratory droplets of all sizes. The term “droplet nuclei” is used to refer to droplets that remain airborne (typically less than 5 μm in diameter).
Each transmission experiment involved eight guinea pigs. On day 0, four of the eight guinea pigs were inoculated intranasally with 10³ PFU of influenza A/Panama/2007/99 virus (150 μl per nostril in phosphate buffered saline [PBS] supplemented with 0.3% bovine serum albumin [BSA]) and housed in a separate room from the remaining animals. At 24 h p.i., each of the eight guinea pigs was placed in a “transmission cage”, a standard rat cage (Ancare R20 series) with an open wire top, which has been modified by replacing one side panel with a wire grid. The transmission cages were then placed into the environmental chamber (Caron model 6030) with two cages per shelf, such that the wire grids opposed each other (Figure 1). In this arrangement, the guinea pigs cannot come into physical contact with each other. Each infected animal was paired on a shelf with a naïve animal. The guinea pigs were housed in this way for 7 d, after which they were removed from the chamber and separated. On day 2 p.i. (day 1 post-exposure) and every second day thereafter up to day 12 p.i., nasal wash samples were collected from anesthetized guinea pigs by instilling 1 ml of PBS-BSA into the nostrils and collecting the wash in a Petri dish. Titers in nasal wash samples were determined by plaque assay of 10-fold serial dilutions on Madin Darby canine kidney cells. Serum samples were collected from each animal prior to infection and on day 17 post-infection, and seroconversion was assessed by hemagglutination inhibition assay.
All transmission experiments reported herein were performed between September 2006 and April 2007.

To test for universality of primers and cycling conditions, we performed parallel experiments in three different laboratories (Berkeley, Cologne, Konstanz) using the same primers but different biochemical products and thermocyclers, and slightly different protocols.
The selected primers for 16S [30 ] amplify a fragment of ca. 550 bp (in amphibians) that has been used in many phylogenetic and phylogeographic studies in this and other vertebrate classes: 16SA-L, 5' - CGC CTG TTT ATC AAA AAC AT - 3'; 16SB-H, 5' - CCG GTC TGA ACT CAG ATC ACG T - 3'.
For COI we tested (1) three primers designed for birds [7 (link)] that amplify a 749 bp region near the 5'-terminus of this gene: BirdF1, 5' - TTC TCC AAC CAC AAA GAC ATT GGC AC - 3', BirdR1, 5' - ACG TGG GAG ATA ATT CCA AAT CCT G - 3', and BirdR2, 5' - ACT ACA TGT GAG ATG ATT CCG AAT CCA G - 3'; and (2) one pair of primers designed for arthropods [2 (link)] that amplify a 658 bp fragment in the same region: LCO1490, 5' - GGT CAA CAA ATC ATA AAG ATA TTG G - 3', and HCO2198, 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3'. Sequences of additional primers for COI that had performed well in mammals and fishes were kindly made available by P. D. N. Hebert (personal communication in 2004) and these primers yielded similar results (not shown).
The optimal annealing temperatures for the COI primers were determined using a gradient thermocycler and were found to be 49–50°C; the 16S annealing temperature was 55°C. Successfully amplified fragments were sequenced using various automated sequencers and deposited in Genbank. Accession numbers for the complete data set of adult mantellid sequences used for the assessment of intra- and interspecific divergences (e.g. in Fig. 5) are AY847959–AY848683. Accession numbers of the obtained COI sequences are AY883978–AY883995.
Nucleotide variability was scored using the software DNAsp [31 (link)] at COI and 16S priming sites of the following complete mitochondrial genomes of nine amphibians and 59 other vertebrates: Cephalochordata: AF098298, Branchiostoma. Myxiniformes: AJ404477, Myxine. Petromyzontiformes: U11880, Petromyzon. Chondrichthyes: AJ310140, Chimaera; AF106038, Raja; Y16067, Scyliorhinus; Y18134, Squalus. Actinopterygii: AY442347, Amia; AB038556, Anguilla; AB034824, Coregonus; M91245, Crossostoma; AP002944, Gasterosteus; AB047553, Plecoglossus; U62532, Polypterus; U12143, Salmo. Dipnoi: L42813, Protopterus. Coelacanthiformes: U82228, Latimeria. Amphibia, Gymnophiona: AF154051, Typhlonectes. Amphibia, Urodela: AJ584639, Ambystoma; AJ492192, Andrias; AF154053, Mertensiella; AJ419960, Ranodon. Amphibia, Anura: AB127977, Buergeria; NC_005794, Bufo; AY158705; Fejervarya; AB043889, Rana; M10217, Xenopus. Testudines: AF069423, NC_000886, Chelonia; Chrysemys; AF366350, Dogania; AY687385, Pelodiscus; AF039066, Pelomedusa. Squamata: NC_005958, Abronia; AB079613, Cordylus; AB008539, Dinodon; AJ278511, Iguana; AB079597, Leptotyphlops; AB079242, Sceloporus; AB080274, Shinisaurus. Crocodilia: AJ404872, Caiman. Aves: AF363031, Anser; AY074885, Arenaria; AF090337, Aythya; AF380305, Buteo; AB026818, Ciconia; AF362763, Eudyptula; AF090338, Falco; AY235571, Gallus; AY074886, Haematopus; AF090339, Rhea; Y12025, Struthio. Mammalia: X83427, Ornithorhynchus; Y10524, Macropus; AJ304826, Vombatus; AF061340, Artibeus; U96639, Canis; AJ222767, Cavia ; AY075116, Dugong; AB099484, Echinops; Y19184, Lama; AJ224821, Loxodonta; AB042432, Mus; AJ001562, Myoxus; AJ001588, Oryctolagus; AF321050, Pteropus; AB061527, Sorex; AF348159, Tarsius; AF217811, Tupaia; AF303111, Ursus (for species names, see Genbank under the respective accession numbers).
16S sequences of a large sample of Madagascan frogs were used to build a database in Bioedit [32 ]. Tadpole sequences were compared with this database using local BLAST searches [33 (link)] as implemented in Bioedit.
The performance of COI and 16S in assigning taxa to inclusive major clades was tested based on gene fragments homologous to those amplified by the primers used herein (see above), extracted from the complete mitochondrial sequences of 68 vertebrate taxa. Sequences were aligned in Sequence Navigator (Applied Biosystems) by a Clustal algorithm with a gap penalty of 50, a gap extend penalty of 10 and a setting of the ktup parameter at 2. PAUP* [34 ] was used with the neighbor-joining algorithm and LogDet distances and excluding pairwise comparisons for gapped sites. We chose these simple phenetic methods instead of maximum likelihood or maximum parsimony approaches because they are computationally more demanding and because the aim of DNA barcoding is a robust and fast identification of taxa rather than an accurate determination of their phylogenetic relationships.

For heat-induced antigen retrieval, 5 µm thick sections were boiled for 30 min at 93 °C in 1 × epitope retrieval solution (Cat# AR9961, Biosystems). The slides were stained overnight at 4 °C with the primary antibody for insulin (Cat# A0564, Agilent), washed twice in PBS (5 min), stained for 2 h at room temperature with a CD45 antibody (Rat Anti-CD45; 30-F11, Cat# 553076, BD Bioscience) and washed twice with PBS (5 min). The secondary antibodies were applied for 2 h at room temperature (Alexa647 goat anti-guinea pig IgG and Alexa555 goat anti-rat IgG; Cat# A-21450 and A-21434, respectively, Thermo Fisher Scientific), washed twice with PBS, before mounting with a fluorescence mounting media (Cat# S3023, Dako). Pictures were acquired using a Nikon microscope at 4× magnification.
Images were analyzed using Fiji software (1.52n with Java 1.8.0_172). Analysis was performed in a semi-automated way; ilastik software (Version 1.3.2, https://www.ilastik.org/) was trained twice, once to recognize pancreas area excluding background and lymph nodes, and the second time to recognize islets. The masks ilastik generated were used to quantify the areas with Fiji (1.52n with Jaca 1.8.0_172). Beta-cell mass was defined as area of insulin positive cells/area of pancreas*weight of the pancreas. Two sections per animal were quantified.

Fully conscious, heparinized animals underwent 50% ET with PolyCHb solution (Hb concentration 6%, human albumin 4%; the PolyCHb and human albumin were mixed immediately before ET). The blood was collected through the arterial catheter, and the PolyCHb solution was transfused through the venous catheter by syringe pumps simultaneously at a rate of 1 mL/min. The total blood volume of a guinea pig was calculated as 0.07 (milliliters per gram) × body weight (grams) (Ancill, 1956 (link)). Immediately after ET, AA injection solution was injected to the body via the venous catheter, by a 1 mL disposable syringe. The dosage of AA is calculated by multiplying the target concentration (2 mmol/L) with the blood volume. Blood samples (300 μL) were obtained from the arterial catheter pre-transfusion together at 0 and 4 h after ET. The guinea pigs were individually housed in metabolism cages for 4 h pre- and post-ET to collect the urine samples. The animals were sacrificed 4 h post-ET and the kidneys were dissected and rinsed with pre-cold saline. One kidney was cut in half and frozen immediately in liquid nitrogen, and the other kidney was fixed in 10% paraformaldehyde.