Material and methods
Field surveys and samplingnext section
We carried out eight field surveys between November 2013 and September 2015 at numerous amphibian habitats over ca. 177 km2 in the Hula Valley. Daytime surveys for L. nigriventer involved searching terrestrial habitats as well as various water bodies where we used dip nets to search for adults, tadpoles and egg clutches. Night surveys only involved visual inspection of water bodies and their banks. To minimise disturbance, sites were usually not inspected more than twice a week. Equipment and shoes were either completely dried or disinfected with Virkon S or 10% bleach solution between locations (i.e. those not connected by waterways) following Johnson et al. (2003). Detailed coordinates of confirmed locations are not published to avoid disturbance and collecting, but have been communicated to the Israel Nature and Parks Authority.
Metamorphosed individuals were captured with gloved hands, photographed in dorsal and ventral views and morphometric measurements were taken. We collected tissue and buccal swabs for assessing genetic variation, and took skin and cloacal swabs for exploring the microbial communities of L. nigriventer and other local amphibians. Skin swabs were also used for Bd screening (Hyatt et al., 2007). Before swabbing, frogs were rinsed with sterilised distilled water to remove transient bacteria (Culp et al., 2007; Lauer et al., 2007; Rebollar et al., 2014). Water volume was adjusted based on SVL (50–150 ml). The skin of each individual was swabbed dorsally and ventrally using two separate swabs with each side receiving 10 strokes.
Tissue samples and buccal swabs were directly stored in 95–99% ethanol, while skin swabs were immediately placed on ice and transferred to freezer storage (–20 °C) within 8 hours. All metamorphosed individuals were released back to their collection site after examination. None were sacrificed but dead individuals (e.g. due to predation, road kills) were preserved in ethanol. Several tadpoles died shortly after capture and were fixed in 70% ethanol and later preserved in 70% ethanol or 5% formalin.
External transmitters (SOPR-2038; Wildlife Materials International, Inc.) were fitted to seven large (> 70 mm) individuals with an elastic waistband. Specimens were kept in a terrarium for up to three days to ensure that the waistband was not causing undue harm. Released individuals were tracked with a TRX-16 receiver and 3-element folding antenna (Wildlife Materials International, Inc.). Tracking was completed twice a day for up to 18 days, but individuals were only visually inspected every second or third day in order to minimise disturbance (Fig. 2 O).
Adult and juvenile morphometrics and colour
In adult and juvenile frogs the following measurements were taken with a calliper to the nearest 0.1 mm, and weight was taken with a digital scale or Pesola spring scale (precision: 0.1 g): horizontal eye diameter (ED), horizontal eye neck fold to snout (FS), hand length (HAL), head width at eyes (HW), interorbital distance (IOD), length of elbow to finger tip (LE), tip of characteristic colour patch on forehead to snout (PS), snout-vent length (SVL), tarsal length (TSL), weight (W), webbing formula is given following Blommers-Schlösser (1979).
We calculated the body condition of each adult individual with the relative mass (W r ) condition index as described in Sztatecsny and Schabetsberger (2005). The obtained ratios were statistically analysed by Mann-Whitney-U test implemented in R (v 3.2.4).
The distinctive ventral and dorsal natural markings displayed by each metamorphosed frog were used for identifying recaptured individuals.
Morphological description and measurements of tadpoles (to the nearest 0.1 mm) were obtained using a stereomicroscope (Zeiss Discovery V12) following landmarks, terminology and definitions of Altig and McDiarmid (1999). Developmental stages were identified following Gosner (1960). Labial tooth row formula (LTRF) follows Altig and McDiarmid (1999) but labial teeth are named ‘keratodonts’. For a list of morphological characters measured and abbreviations used, see tadpole description in Appendix 3.
Calls probably representing advertisement calls were recorded in February 2015 from two adult males (SVL 114 and 121 mm) kept in a terrarium (L × W × H = 71 × 50.5 × 40 cm; filled with water (7 cm depth), with some reed and grass material) overnight together with two females (SVL 103 and 128 mm). Calls were recorded above water with the built-in microphone of a waterproof camera (Pentax; WG-II) and an acoustic recorder (SM2+; Wildlife Acoustics Inc.) that also recorded air temperature. Water temperature was measured by a Thermochron iButton datalogger. Recordings were analysed at a sampling rate of 44.1 kHz and 16-bit resolution with Cool Edit Pro 2.0. We measured interval length between calls and six acoustic features for each call, including call duration, expiratory note duration, inspiratory note duration, dominant frequency of the total call, and of presumed expiratory and inspiratory notes (Glaw and Vences, 1991). Vocalisations were illustrated using the R package ‘seewave’ (Sueur et al., 2008). Call recordings were submitted to the FonoZoo sound archive (http://www.fonozoo.org); accession numbers 9850–9854.
Bacterial communities and Bd/Bsal screening
A screening for the two chytridiomycete pathogens, Bd and B. salamandrivorans Martel, Blooi, Bossut and Pasmans, 2013 (Bsal), was conducted in L. nigriventer, Pelophylax bedriagae (Camerano, 1882), Hyla savignyi Audouin, 1827 and Salamandra infraimmaculata Martens, 1885 from seven localities in and around the Hula Valley, to identify the possible presence or absence of these two amphibian pathogens from this region in Israel. Skin microbial communities were analysed for L. nigriventer and the syntopic Levant water frogs (P. bedriagae).
DNA was extracted from swabs with the PowerSoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Ca, USA), following the Earth Microbiome project protocol (http://www.earthmicrobiome.org) except that centrifuge conditions were adjusted to accommodate reduced rotor speed.
Bd/Bsal screening was performed by duplex quantitative PCR (in duplicate) according to the protocol of Blooi et al. (2013).
To characterise skin bacterial communities, we PCR-amplified the V4 region of the bacterial 16S rRNA gene with dual-indexed primers (Kozich et al., 2013). PCRs were completed in duplicate following Sabino-Pinto et al. (2016). Negative controls were included to check for contamination. PCR products were combined for each sample and roughly quantified on 1% agarose gels. Approximately equal concentrations of PCR amplicons from each sample were pooled, gel purified using the MinElute gel extraction kit (Qiagen). DNA concentration was determined using a Qubit 2.0 and equimolar amounts were sequenced on an Illumina MiSeq platform at the Helmholtz Center for Infection Research in Braunschweig, Germany, using paired-end 2x250 v2 chemistry. Sequences were deposited in the NCBI Short Read Database (SRA BioProject PRJNA326938).
Sequences were processed with the Quantitative Insights Into Microbial Ecology (QIIME; v 1.9.1.) pipeline for Linux (Caporaso et al., 2010). Raw forward and reverse reads of each sample were joined using Fastq-join under default settings (Aronesty, 2011, 2013). After quality filtering with default settings to remove low-quality sequences, we further filtered the reads by length (250–253 bp; usegalaxy.org) and removed chimeric sequences on a per sample basis using de novo usearch61 chimera detection within QIIME (http://drive5.com/usearch/usearch_docs.html; Edgar et al., 2011). Sequences were clustered into bacterial operational taxonomic units (OTUs) with a sequence similarity threshold of 97% using an open reference OTU-picking strategy (Rideout et al., 2014, http://qiime.org/tutorials/open_reference_illumina_processing.html). SILVA 119 (24 July 2014 release; https://www.arb-silva.de) served as reference database and UCLUST (Edgar, 2010) was used in the de novo clustering steps. The most abundant sequences of each OTU were selected as representative sequences and aligned using PyNAST (Caporaso et al., 2010). OTUs with less than 0.005% of total reads were removed from the data set following Bokulich et al. (2013). Taxonomy was assigned using the RDP classifier (Wang et al., 2007) with the SILVA 119 taxonomy and representative sequences as reference, and a phylogenetic tree built using FastTree (Price et al., 2010) adhering to QIIME’s standard procedures. We also used QIIME to generate a rarefaction curve to confirm that an asymptote was reached for all samples (see S1 in the Supplement). Lastly, we rarefied the data to 1000 sequences to correct for sample depth heterogeneity and calculated beta diversity using the weighted UniFrac distance metric in QIIME.
We tested for (i) differences in dorsal and ventral skin bacterial communities of L. nigriventer, (ii) seasonal variation of the skin microbial community, and (iii) species-specific differences between L. nigriventer and P. bedriagae. Statistical analyses were done with PRIMER v7 software (Plymouth Routines In Multivariate Ecological Research; Clarke and Gorley, 2015). PERMANOVA analyses were performed using 999 permutations and associated plots were generated by principal coordinates analysis (PCoA). Core bacterial communities, defined as OTUs present in at least 75% of the samples and overlap among core communities was visualised with Venn diagrams drawn with VENNY 2.0 (http://bioinfogp.cnb.csic.es/tools/venny/).
Skin peptides collection and anti-Bd growth inhibition assays
Skin secretions of two L. nigriventer individuals were collected from two sterile polyethylene bags (Whirl-Pak, Nasco) in which they were kept prior to measurement, by washing the bags with ~50 ml of sterile water. Samples were then frozen until further processing. After defrosting, 2 ml of the skin secretion wash was removed for direct testing against Bd. The remaining volume from each sample was acidified to a final volume of 1% HCL to inactivate potential endogenous peptidases (Resnick et al., 1991; Steinborner et al., 1997) and immediately loaded onto C-18 Sep-Pak cartridges (Waters Corporation, Milford, Massachusetts, USA) which were then stored in vials with 2–5 ml of 0.1% HCL until further processing.
We eluted the peptides bound to the Sep-Paks with 70% acetonitrile, 29.9% water, and 0.1% trifluoracetic acid (v/v/v) and centrifuged them under vacuum to concentrate them to dryness. After Sep-Pak purification, we determined the total concentration of the recovered skin peptides by Micro BCA Assay (Pierce, Rockford, Illinois, USA) following manufacturer’s instructions, except that we used bradykinin (RPPGFSPFR; Sigma) to establish a standard curve (Rollins-Smith et al., 2002). Skin peptides were analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) as described in Pask et al. (2012). We analysed each sample by averaging signals from 250 consecutive laser shots. Mass spectrometry data was acquired in the mass/charge (m/z) range 500 to 7,000, truncated at m/z 4,000 and baseline-corrected with Data Explorer v4.4 (Applied Biosystems). The peak values shown represent the monoisotopic mass, [M+H]+. A few signals may show secondary peaks 22 mass units greater than the primary peak and probably represent a peptide plus sodium adduct [M+Na]+. Spectra may also show peaks at m/z 568.1 and 650.0 because of matrix or background signal.
We conducted in vitro growth inhibition assays of the enriched skin peptide mixtures against two Bd isolates (JEL 197 and ‘Section Line’) (Longcore et al., 1999; Piovia-Scott et al., 2015) as described previously (Rollins-Smith et al., 2006; Ramsey et al., 2010; Holden et al., 2015). Briefly, B. dendrobatidis zoospores were grown on 1% tryptone agar for one week at 23°C. Freshly isolated zoospores were added (5 × 104/50 µl, 5 replicates) in tryptone broth to a 96-well flat-bottom microtiter plates with 50 µl of a serially diluted mixture of skin peptides dissolved in HPLC-grade water. Positive control wells contained zoospores and 50 µl HPLC water. Negative control wells contained heat-killed zoospores (60°C for 10 minutes) and 50 µl of HPLC water. Plates were incubated at 23°C for one week, and growth was measured as an increased optical density at 490 nm (OD490 ) using an MRX Microplate Reader (Dynex Technologies, Inc., Chantilly, VA, USA). Percent growth was calculated as follows: OD at Day 7 – OD at Day 0 for test sample / OD at Day 7 – OD at Day 0 for positive control.
We performed Bd growth inhibition assays with the direct skin secretion solution following the methods explained above, with 50 µl of the direct skin secretion solution being added to the microtiter plate instead of the peptide dilutions. The assays were completed with the isolate Bd VMV 813.