Transcriptome analyses of immune tissues from three Japanese frogs (genus Rana ) reveals their utility in characterizing major histocompatibility complex class II

Abstract

BackgroundIn Japan and East Asia, endemic frogs appear to be tolerant or not susceptible to chytridiomycosis, a deadly amphibian disease caused by the chytrid fungus Batrachochytridium dendrobatidis (Bd). Japanese frogs may have evolved mechanisms of immune resistance to pathogens such as Bd. This study characterizes immune genes expressed in various tissues of healthy Japanese Rana frogs.ResultsWe generated transcriptome data sets of skin, spleen and blood from three adult Japanese Ranidae frogs (Japanese brown frog Rana japonica, the montane brown frog Rana ornativentris, and Tago’s brown frog Rana tagoi tagoi) as well as whole body of R. japonica and R. ornativentris tadpoles. From this, we identified tissue- and stage-specific differentially expressed genes; in particular, the spleen was most enriched for immune-related genes. A specific immune gene, major histocompatibility complex class IIB (MHC-IIB), was further characterized due to its role in pathogen recognition. We identified a total of 33 MHC-IIB variants from the three focal species (n = 7 individuals each), which displayed evolutionary signatures related to increased MHC variation, including balancing selection. Our supertyping analyses of MHC-IIB variants from Japanese frogs and previously studied frog species identified potential physiochemical properties of MHC-II that may be important for recognizing and binding chytrid-related antigens.ConclusionsThis is one of the first studies to generate transcriptomic resources for Japanese frogs, and contributes to further understanding the immunogenetic factors associated with resistance to infectious diseases in amphibians such as chytridiomycosis. Notably, MHC-IIB supertyping analyses identified unique functional properties of specific MHC-IIB alleles that may partially contribute to Bd resistance, and such properties provide a springboard for future experimental validation.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4404-0) contains supplementary material, which is available to authorized users.

Peer Review #3 of "Selective constraint acting on TLR2 and TLR4 genes of Japanese Rana frogs (v0.2)"

“…To isolate TLR2 and TLR4 genes, we utilised the published transcriptomic data set previously compiled using Illumina sequences from immune tissues of the three species (Lau et al, 2017). Briefly, we used the assembled transcripts that were annotated with NCBI-BLAST-2.3.30 against the Swissprot protein database (httpr://www.expasy.ch/sprot), and isolated all transcripts that had top hits from BLAST search to published TLR genes of other vertebrates.…”

“…We then compared the d N /d S ratio with that of major histocompatibility complex (MHC) class I and II (average among the three species), which are known to be under balancing selection in these species (Lau et al, 2016(Lau et al, , 2017. In addition, we compared with d N /d S ratio of all orthologous genes collated from the transcriptome data set of Lau et al (2017). This consisted of over 3000 orthologous amino acid sequences from each of the three species that were identified using Proteinortho V5.15 (Lechner et al, 2011).…”

“…In order to investigate baseline expression of our candidate TLRs, we extracted expression levels from our transcriptome data set (Lau et al, 2017). RSEM v 1.3.0 (Li & Dewey, 2011) was used to extract trimmed mean log expression ratio, or TMM-normalized values which represent estimated relative RNA production levels (Robinson & Oshlack, 2010), for TLR2 and TLR4 transcripts in each of the 12 samples (Table S1).…”

“…In addition, one PSS each of R. tagoi tagoi (T128) and R. ornativentris (Q253) corresponded to human TLR4 sites predicted to be involved in secondary (N268) and phosphate (K388) binding, respectively, of bacterial lipopolysaccharides (Park et al, 2009). Positive selection at identical codon sites across different species, as observed in MHC adaptive immune genes (Lau et al, 2016(Lau et al, , 2017, could be driven by a single selective force that is pathogen-related.…”

“…While adaptive immune genes of Japanese frogs have been studied in the context of diseases like chytridiomycosis (Lau et al, 2016(Lau et al, , 2017, there are limited studies involving innate immunity genes including TLRs. Since TLR2 and TLR4 have been shown to play a role in innate immune responses to various fungi (Roeder et al, 2004;Luther & Ebel, 2006), TLRs therefore may be involved in Bd resistance (Richmond et al, 2009 Manuscript to be reviewed measured using with quantitative RT-PCR, were variable between different adult tissues (Zhao et al, 2014).…”