Contributions to Zoology, 86 (1) – 2017R.G. Bina Perl; Sarig Gafny; Yoram Malka; Sharon Renan; Douglas C. Woodhams; Louise Rollins-Smith; James D. Pask; Molly C. Bletz; Eli Geffen; Miguel Vences: Natural history and conservation of the rediscovered Hula painted frog, Latonia nigriventer
Results

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Bd/Bsal screening, skin bacterial community, and defensive skin peptides

The pathogen Bd was detected in 32% of the tested amphibian individuals (n = 87) from northern Israel, while none were positive for Bsal. We found Bd in two amphibian species (L. nigriventer and P. bedriagae) and in three of the seven examined locations within the Hula Valley (Hula Nature Reserve, Kiryat Shmona and Yesod HaMa’ala). Infection loads for Bd-positive individuals ranged between 1–311 genomic equivalents of zoospores per swab (Table 4).

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Table 4. Number of individuals per species tested for Batrachochytrium dendrobatidis including mean (range) of genomic equivalents of zoospores per swab for positive tested specimen.

Bacterial communities of L. nigriventer were comprised of Proteobacteria (56.7%) with a high representation of Gammaproteobacteria (33.4% of the overall community), Bacteroidetes (25.3%) and Firmicutes (6.7%) (Fig. 9 A). The 20 most abundant OTUs found on the skin of L. nigriventer represented 42% of the total reads (Fig. 9 B; S6 in the Supplement). The most abundant OTU (7% of the total sequences) was assigned to an unspecified Chryseobacterium and present in 100% of the samples, although in varying abundance (< 1–24% of the reads). Comparisons based on weighted UniFrac distances did not reveal significant differences between (i) microbial communities from the ventral versus dorsal skin of L. nigriventer (PERMANOVA: N = 27; p = 0.117; Fig. 10 A), (ii) ventral surfaces of females versus males (PERMANOVA: N = 15; p = 0.646; Fig. 10 B) or (iii) ventral surfaces of Bd-positive versus Bd-negative individuals (PERMANOVA: N = 22; p = 0.283; Fig. 10 C). However, we observed a significant shift in the ventral skin microbial community over time.

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Fig. 9. Relative abundances of major bacterial taxa obtained from Latonia nigriventer skin samples in Yesod HaMa’ala as identified by the SILVA 119 database. The order of the taxa in columns corresponds to that in legends (ordered alphabetically). A) Abundances of dominant bacterial phylotypes; B) abundances of the 20 most frequent bacterial OTUs. (For detailed OTU IDs see S5 in the Supplement).

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Fig. 10. Principal coordinates analysis plots of weighted UniFrac distances of the microbial communities associated with Latonia nigriventer and Pelophylax bedriagae. A) Comparison of dorsal and ventral surfaces in L. nigriventer; B) comparison of ventral skin microbial communities found in female and male L. nigriventer; C) comparison of L. nigriventer individuals tested positive and negative for chytrid (ventral surfaces only); D) seasonal changes in the ventral skin microbial community associated with L. nigriventer; E) comparison of the ventral skin microbial communities found in P. bedriagae and L. nigriventer; F) Venn diagram depicting the overlap of core microbial communities as obtained from ventral skin swabs of L. nigriventer and P. bedriagae captured at the same location and day. The minimum fraction of samples an OTU must be observed in was set to 75%.

While no significant changes were observed between the ventral skin samples taken in mid-February and mid-April (N = 13; p = 0.799) or between those collected in mid-April and the end of June (N = 15; p = 0.0.093), significant differences were detected for all other time-associated comparisons: mid-February – late June (N = 15; p = 0.012); mid-February – mid-September (N = 15; p = 0.001); late June – mid-September (N = 17; p = 0.001) (Fig. 10 D).

The results obtained for the skin-associated bacterial communities of syntopic P. bedriagae were similar to those of L. nigriventer: no significant differences between ventral versus dorsal surfaces of the same individuals (N = 14; p = 0.898) nor between ventral surfaces of Bd-positive versus Bd-negative individuals (N = 22; p = 0.366).

A comparison of the ventral skin-associated communities of L. nigriventer and P. bedriagae from the same location and same time-point revealed differences between the two species (N = 17; p = 0.001; Fig. 10 E). The core bacterial communities contained 30 OTUs (88% of the core skin microbiota of L. nigriventer and 57% of that of P. bedriagae) that were present on the ventral skin of at least 75% of the individuals of both species (Fig. 10 F).

The skin secretions collected from two different individuals and examined for peptide composition had significant amounts of hydrophobic peptides recovered after C18 enrichment. We detected a number of common peptide mass signals shared by both frog individuals. The mass ranges are suggestive of possible antimicrobial peptides (Table 5; S7 in the Supplement). In a growth inhibition assay, the mixture of peptides inhibited the growth of two different Bd isolates (JEL 197 and ‘Section Line’; Fig. 11). At the highest concentration tested (500 μg/ml), Bd growth inhibition ranged from 51% to 91.5% against the Section Line isolate and 70–82% inhibition against the original type isolate JEL 197. Both isolates are among the global panzootic lineages (Schloegel et al., 2012; Piovia-Scott et al., 2015). Furthermore, the direct skin secretion solution was found to inhibit Bd by 35–36%.

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Table 5. Quantity of peptides detected in the mucus of two Latonia nigriventer females.

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Fig. 11. Bd growth inhibition assays of two female Latonia nigriventer individuals against two Bd strains. A) Female 1 (SVL 90 mm; weight 86 g) against Bd 197; B) female 2 (SVL 128 mm; weight 187 g) against Bd 197; C) female 1 against Bd Section Line strain; D) female 2 against Bd Section Line strain. Asterisks (*) indicate significantly less than positive control for growth by Student’s t test (p < 0.05).