The Unique Antimicrobial Recognition and Signaling Pathways in Tardigrades with a Comparison Across Ecdysozoa

Abstract

Tardigrades are microscopic animals known to withstand unfavorable abiotic conditions. These animals are also constantly exposed to biotic stresses, including parasites and internal microbiomes. However, the tardigrade immune mechanisms against these biotic stresses are largely uncharacterized. Due to the contentious phylogenetic position of tardigrades, it is not intuitive whether they possess an immune system more similar to that of arthropods (e.g., Toll, Imd, and JNK pathways of the Drosophila melanogaster antimicrobial response) or to that of nematodes (e.g., the Tir-1/Nsy-1/Sek-1/Pmk-1/Atf-7 signaling cassette [called Tir-1 pathway here]) in Caenorhabditis elegans). In this study, comparative genomic analyses were conducted to mine homologs of canonical D. melanogaster and C. elegans immune pathway genes from eight tardigrades (Echiniscoides cf. sigismundi, Echiniscus testudo, Hypsibius exemplaris, Mesobiotus philippinicus, Milnesium tardigradum, Paramacrobiotus richtersi, Richtersius cf. coronifer, and Ramazzottius varieornatus) and four non-arthropod ecdysozoans (two onychophorans: Epiperipatus sp. and Opisthopatus kwazululandi; one nematomorph: Paragordius varius; and one priapulan: Priapulus caudatus) in order to provide insights into the tardigrade antimicrobial system. No homologs of the intracellular components of the Toll pathway were detected in any of the tardigrades examined. Likewise, no homologs of most of the Imd pathway genes were detected in any of the tardigrades or any of the other non-arthropod ecdysozoans. Both the JNK and Tir-1 pathways, on the other hand, were found to be conserved across ecdysozoans. Interestingly, tardigrades had no detectable homologs of NF-κB, the major activator of antimicrobial response gene expression. Instead, tardigrades appear to possess NF-κB distantly related NFAT homologs. Overall, our results show that tardigrades have a unique gene pathway repertoire that differs from that of other ecdysozoans. Our study also provides a framework for future studies on tardigrade immune responses.

Keywords: Genetics of Immunity; biotic stress response; ecdysozoan immune response; tardigrade immune response.

Figure 1

Tardigrade and other ecdysozoan Toll pathway gene homologs. (A) The Drosophila melanogaster Toll pathway. (B) The presence (filled boxes) and absence (empty boxes) of D. melanogaster Toll pathway gene homologs in tardigrades and other ecdysozoans. LRR – extracellular leucine rich repeat domain; Mid – transmembrane domain; TIR – intracellular domain.

Figure 2

Tardigrade and other ecdysozoan Toll-like receptor homologs. (A) Predicted structure of TIR-containing D. melanogaster Toll receptor homologs of tardigrades and other ecdysozoans. (B) Phylogenetic tree of TIR domains of the Toll receptor homologs of tardigrades, other ecdysozoans, and vertebrates built using the LG+G model with 1000 bootstrap replicates and midpoint-rooted. Complete bootstrap values available in Figure S1. Branch colors represent different phyla. Scale bar = number of nucleotide substitution/site.

Figure 3

Tardigrade and other ecdysozoan Imd pathway gene homologs. (A) The Drosophila melanogaster Imd pathway. (B) The presence (filled boxes) and absence (empty boxes) of D. melanogaster Imd pathway gene homologs in tardigrades and other ecdysozoans. Ext PGRP – extracellular PGRP; Tr PGRP – transmembrane PGRP; Ext – extracellular domain; Mid – transmembrane domain; Int – intracellular domain; Ank – ankyrin repeats.

Figure 4

Phylogenetic tree of Rel homology domain (RHD)-containing proteins in the 1^st set of candidate genes of tardigrades and non-arthropod ecdysozoans, together with Dif, Dorsal, Relish, and NFAT orthologs of poriferans, cnidarians, arthropods, mollusks, annelids, echinoderms, and chordates. The tree was built using the VT+G+I model with 1000 bootstrap replicates and midpoint rooted. Branch colors represent different phyla. Complete bootstrap values available in Figure S2. Scale bar = number of nucleotide substitution/site.

Figure 5

Tardigrade and other ecdysozoan JNK pathway gene homologs. (A) The Drosophila melanogaster JNK pathway. (B) The presence (filled boxes) and absence (empty boxes) of D. melanogaster JNK pathway gene homologs in tardigrades and other ecdysozoans.

Figure 6

Tardigrade and other ecdysozoan Tir-1 pathway gene homologs. (A) The Caenorhabditis elegans Tir-1 pathway. (B) The presence (filled boxes) and absence (empty boxes) of C. elegans Tir-1 pathway gene homologs in tardigrades and other ecdysozoans.