Transcriptome analysis of air-breathing land slug, Incilaria fruhstorferi reveals functional insights into growth, immunity, and reproduction

Abstract

Background: Incilaria (= Meghimatium) fruhstorferi is an air-breathing land slug found in restricted habitats of Japan, Taiwan and selected provinces of South Korea (Jeju, Chuncheon, Busan, and Deokjeokdo). The species is on a decline due to depletion of forest cover, predation by natural enemies, and collection. To facilitate the conservation of the species, it is important to decide on a number of traits related to growth, immunity and reproduction addressing fitness advantage of the species.

Results: The visceral mass transcriptome of I. fruhstorferi was enabled using the Illumina HiSeq 4000 sequencing platform. According to BUSCO (Benchmarking Universal Single-Copy Orthologs) method, the transcriptome was considered complete with 91.8% of ortholog genes present (Single: 70.7%; Duplicated: 21.1%). A total of 96.79% of the raw read sequences were processed as clean reads. TransDecoder identified 197,271 contigs that contained candidate-coding regions. Of a total of 50,230 unigenes, 34,470 (68.62% of the total unigenes) annotated to homologous proteins in the Protostome database (PANM-DB). The GO term and KEGG pathway analysis indicated genes involved in metabolism, phosphatidylinositol signalling system, aminobenzoate degradation, and T-cell receptor signalling pathway. Many genes associated with molluscan innate immunity were categorized under pathogen recognition receptor, TLR signalling pathway, MyD88 dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related. The reproduction-associated unigenes showed homology to protein fem-1, spermatogenesis-associated protein, sperm associated antigen, and testis expressed sequences, among others. In addition, we identified key growth-related genes categorized under somatotrophic axis, muscle growth, chitinases and collagens. A total of 4822 Simple Sequence Repeats (SSRs) were also identified from the unigene sequences of I. fruhstorferi.

Conclusions: This is the first available genomic information for non-model land slug, I. fruhstorferi focusing on genes related to growth, immunity, and reproduction, with additional focus on microsatellites and repeating elements. The transcriptome provides access to greater number of traits of unknown relevance in the species that could be exploited for in-depth analyses of evolutionary plasticity and making informed choices during conservation planning. This would be appropriate for understanding the dynamics of the species on a priority basis considering the ecological, health, and social benefits.

Keywords: Immunity; Incilaria fruhstorferi; Peptidoglycan recognition protein; Sex-linked genes; Simple sequence repeats; Tollip; Transciptome; de novo analysis.

Fig. 1

Size distribution of I. fruhstorferi (a) visceral mass assembled sequences. The clean reads were clustered using the Trinity de novo assembly, redundancy from the clustered sequences were removed using the TransDecoder program, defining the potential coding regions. (b) Contig length distribution, (c) Non-redundant sequences length distribution, (d) unigene length distribution

Fig. 2

Annotation of I. fruhstorferi unigenes against the public protein and nucleotide databases. Venn diagram showing homologous matches of unigenes to PANM DB, UniGene, SwissProt, and KOGDB (either specific or overlapping)

Fig. 3

KOG functional classification of I. fruhstorferi unigenes. Of the 50,230 non-redundant unigene sequences, 22,984 classified under 25 functional KOG categories excluding the multifunctional category. Predominantly, the unigenes classified under R- General function and Multi category

Fig. 4

GO classification of I. fruhstorferi unigenes at level 2. The unigene sequences classified under different functional categories under the GO term notation of (a) Molecular Function, (b) Cellular Component, and (c) Biological Process

Fig. 5

Distribution of Simple Sequence Repeat (SSR) types found in the unigenes of I. fruhstorferi. The most predominant repeat types included the dinucleotides AT/AT and AC/GT

Fig. 6

Phylogenetic relationships inferred for IfTollip using the maximum-likelihood method of MEGA7 program. The evolutionary distances were computed using the Poisson correction method. The tree was bootstrapped (1000 replications) and the values are indicated by numbers at nodes. GenBank accession numbers downloaded from Ensembl and NCBI are: HsTollip, Q9H0E2.1; MmTollip, Q9QZ06; GgTollip, F1P006; XlTollip, Q3B8H2;AjTollip, AHA83602.1; AgTollip, XP_562476; MjTollip, BAK19511; LvTollip, AET79206; CgTollip, EKC34473; MgTollip AHI17285; CeTollip, NP_492757; SpTollip, XP_001196148; DrTollip, AAH46009.1; PyTollip, AK062848.1; TsTollip, XP_003379806.1; BgTollip, XP_013073937.1 and AcTollip XP_012936924.1

Fig. 7

Phylogenetic analysis of If_PGRP_SC-2. The PGRP domain was considered for the analysis using the maximum likelihood method of MEGA7 program. The tree was bootstrapped (1000 replications) and the values are indicated by numbers at the nodes. The GenBank accession numbers are:: Pa_PGRP, No. JF831447.1; Bg_SC, No. XM_013209309.1; Sg_PGRP, No. JN642118.1; Hd_PGRP, No. KF554145.1; Ec_PGRP2, No. AY956812.1; Hc_S2, No. KF941201.1; AfS1_PGRP, No. AY987008.1; Cg_SC-2, No. XM_011424461.2; Mg_PGRP2, No. KP125936.1 and Dm_SA, No. AF207540.1

Fig. 8

The schematic representation of TLR and JAK-STAT pathways postulated in the slug I. fruhstorferi. IFNR, interferon receptor; JAK, janus kinase; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; IRF, interferon regulatory factor; SR, scavenger receptors; FREP, fibrinogen-related protein; PGRP, peptidoglycan recognition protein; IRAK, interleukin-1 receptor-associated kinase; TRAF, tumor necrosis factor receptor associated factor; ECSIT, evolutionarily conserved signaling intermediate in Toll pathway; TAB, tumor growth factor-beta-activated kinase; TBK, TANK-binding kinase; IKK, IκB kinase; TAK, transforming growth factor-beta activated kinase; NF-κB, nuclear factor kappa B; AP, activator protein