Sex Reversal and Performance in Fitness-Related Traits During Early Life in Agile Frogs

Sex reversal is a mismatch between genetic sex (sex chromosomes) and phenotypic sex (reproductive organs and secondary sexual traits). It can be induced in various ectothermic vertebrates by environmental perturbations, such as extreme temperatures or chemical pollution, experienced during embryonic or larval development. Theoretical studies and recent empirical evidence suggest that sex reversal may be widespread in nature and may impact individual fitness and population dynamics. So far, however, little is known about the performance of sex-reversed individuals in fitness-related traits compared to conspecifics whose phenotypic sex is concordant with their genetic sex. Using a novel molecular marker set for diagnosing genetic sex in agile frogs (Rana dalmatina), we investigated fitness-related traits in larvae and juveniles that underwent spontaneous female-to-male sex reversal in the laboratory. We found only a few differences in early life growth, development, and larval behavior between sex-reversed and sex-concordant individuals, and altogether these differences did not clearly support either higher or lower fitness prospects for sex-reversed individuals. Putting these results together with earlier findings suggesting that sex reversal triggered by heat stress may be associated with low fitness in agile frogs, we propose the hypothesis that the fitness consequences of sex reversal may depend on its etiology.

Exposure to Batrachochytrium dendrobatidis affects chemical defences in two anuran amphibians, Rana dalmatina and Bufo bufo

Background Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide amphibian biodiversity loss. Many amphibians exhibit skin-based chemical defences, which may play an important role against invading pathogens, but whether the synthesis of these chemical compounds is enhanced or suppressed in the presence of pathogens is largely unknown. Here we investigated direct and indirect effects of larval exposure to the globally distributed and highly virulent Bd-GPL strain on skin secreted chemical defences and life history traits during early ontogeny of agile frogs (Rana dalmatina) and common toads (Bufo bufo). Results Exposure to Bd during the larval stage did not result in enhanced synthesis of the antimicrobial peptide Brevinin-1 Da in R. dalmatina tadpoles or in increased production of bufadienolides in B. bufo tadpoles. However, exposure to Bd during the larval stage had a carry-over effect reaching beyond metamorphosis: both R. dalmatina and B. bufo froglets contained smaller quantities of defensive chemicals than their Bd-naïve conspecifics in the control treatment. Prevalence of Bd and infection intensities were very low in both larvae and metamorphs of R. dalmatina, while in B. bufo we observed high Bd prevalence and infection intensities, especially in metamorphs. At the same time, we did not find a significant effect of Bd-exposure on body mass or development rate in larvae or metamorphs in either species. Conclusions The lack of detrimental effect of Bd-exposure on life history traits, even parallel with high infection intensities in the case of B. bufo individuals, is surprising and suggests high tolerance of local populations of these two species against Bd. However, the lowered quantity of defensive chemicals may compromise antimicrobial and antipredatory defences of froglets, which may ultimately contribute to population declines also in the absence of conspicuous mass-mortality events.

Diversity and substrate-specificity of green algae and other micro-eukaryotes colonizing amphibian clutches in Germany, revealed by DNA metabarcoding

Amphibian clutches are colonized by diverse but poorly studied communities of micro-organisms. One of the most noted ones is the unicellular green alga, Oophila amblystomatis, but the occurrence and role of other micro-organisms in the capsular chamber surrounding amphibian clutches have remained largely unstudied. Here, we undertook a multi-marker DNA metabarcoding study to characterize the community of algae and other micro-eukaryotes associated with agile frog (Rana dalmatina) clutches. Samplings were performed at three small ponds in Germany, from four substrates: water, sediment, tree leaves from the bottom of the pond, and R. dalmatina clutches. Sampling substrate strongly determined the community compositions of algae and other micro-eukaryotes. Therefore, as expected, the frog clutch-associated communities formed clearly distinct clusters. Clutch-associated communities in our study were structured by a plethora of not only green algae, but also diatoms and other ochrophytes. The most abundant operational taxonomic units (OTUs) in clutch samples were taxa from Chlamydomonas, Oophila, but also from Nitzschia and other ochrophytes. Sequences of Oophila “Clade B” were found exclusively in clutches. Based on additional phylogenetic analyses of 18S rDNA and of a matrix of 18 nuclear genes derived from transcriptomes, we confirmed in our samples the existence of two distinct clades of green algae assigned to Oophila in past studies. We hypothesize that “Clade B” algae correspond to the true Oophila, whereas “Clade A” algae are a series of Chlorococcum species that, along with other green algae, ochrophytes and protists, colonize amphibian clutches opportunistically and are often cultured from clutch samples due to their robust growth performance. The clutch-associated communities were subject to filtering by sampling location, suggesting that the taxa colonizing amphibian clutches can drastically differ depending on environmental conditions.

Characterisation of the S1 satellite DNA family in Rana dalmatina

In this study, the DNA of Rana dalmatina was digested with Asp 718I and the two bands of highly repeated DNA produced were cloned and characterised. The largest fragment (494 bp) corresponded to the entire repetitive unit of the major satellite DNA (RdS1a), while the smaller fragment of 385 bp corresponded to the major fragment of RdS1a produced by digestion. A fragment of 332 bpbcorresponding to the repetitive unit of satellite S1b (RdS1b) was instead achieved by digestion with Eco RV. RdS1b is highly homologous to the corresponding portion of the repetition of RdS1a and presents the first 36 bp repeated and inverted. This suggested that RdS1b would have been derived from satellite S1a by two distinct and subsequent events. Further, the high sequence homology and length between RdS1a and the S1a of Rana italica (RiS1a) confirmed the hypothesis that the satellite S1a is antecedent to S1b and inherited from a common ancestor. Southern blots of R. dalmatina genomic DNA digested with Asp 718I produced hybrid bands of fragments of different sizes containing in addition to the satellite S1a, also one or more copies of the S1b satellites. The only sequenced band at the moment corresponded to the repetitive unit of the satellite RdS1a + b (826 bp) deleted of the fragment Asp 718I less than RdS1a (109 bp), while the other double bands should almost certainly correspond to repetitive units of satellites RdS1a + 2b and RdS1a + 3b. Our data suggested different satellite DNA organisation in R. dalmatina, including the tandem structure of the repetitive units of the RdS1a or RdS1b. Our data also suggested the existence in R. dalmatina of at least four different types of hybrid repeating units in all the populations examined.

Annotated checklist of the herpetofauna (Amphibia, Reptilia) of Lefkada Island (Ionian Islands, Greece)

The island of Lefkada is a popular and easily accessible tourist destination; nevertheless, there is no comprehensive checklist of its interesting herpetofauna. Indeed, most records are old, scattered in different papers, and lacking precise locality data. In order to summarize the knowledge on the herpetofauna of the island, we critically reviewed literature, we looked for reliable records in the web and we carried out a field survey in May 2019. The herpetofauna of Lefkada amounts to 28 species (six amphibians and 22 reptiles). The occurrence of Testudo graeca on the island is considered unlikely, while an additional species, Testudo marginata, is here reported based on web sources. Among the species recorded on the island, 25 have been recently confirmed, two of them (Rana dalmatina and Anguis graeca) over a century. For three species (Lissotriton graecus, Chelonia mydas, and Podarcis tauricus) the last records date back to over 30 years ago.

Invasive crayfish does not influence spawning microhabitat selection of brown frogs

Microhabitat selection is a key component of amphibian breeding biology and can be modulated in response to the features of breeding sites and the presence of predators. Despite invasive alien species being among the major threats to amphibians, there is limited information on the role of invasive species in shaping amphibians’ breeding microhabitat choice. The invasive red swamp crayfish (Procambarus clarkii) is a major predator of amphibians’ larvae, including those of the brown frogs Rana dalmatina and Rana latastei. Although qualitative information about the spawning site preferences and breeding microhabitat choice of brown frogs is available in the literature, only a few studies performed quantitative analyses, and the relationship between microhabitat choice and the presence of alien predators has not been investigated yet. The aims of this study were: (1) to characterize the microhabitats selected for clutch deposition by R. dalmatina and R. latastei and (2) to test if the position and the aggregation of egg clutches differ in sites invaded or not invaded by P. clarkii. During spring 2017, we surveyed multiple times 15 breeding sites of both brown frogs in Northern Italy; in each site we assessed the features of the microhabitat where each egg clutch was laid, considering its position (distance from the shore, depth of the water column) and the degree of aggregation of clutches. In each site we also assessed the presence/absence of the invasive crayfish and the relative abundance in the breeding period. We detected egg clutches in all sites; the crayfish occurred in eight ponds. Our results showed substantial differences between the spawning microhabitat features of the two brown frogs: Rana latastei clutches showed a higher degree of aggregation and were associated with deeper areas of the ponds , while Rana dalmatina deposited more spaced out clutches in areas of the ponds that were less deep. For both species, spawning microhabitat features were not significantly different between sites with and without P. clarkii. Although we did not detect behavioural responses to P. clarkii in the choice of spawning microhabitat , additional studies are required to assess whether these frogs modulate other behavioural traits (e.g. during larval development) in response to the invasive predator.