Injury-Induced Innate Immune Response During Segment Regeneration of the Earthworm, Eisenia andrei

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.


General histochemistry
Cryostat sections (8 µm) were dried overnight and then hematoxylin-eosin (H&E) staining was applied according to the standard procedure. To detect polysaccharides, sections were incubated for 10 min in 1% periodic acid, followed by incubation in Schiff's (PAS) reagent for 10 min as we described earlier [1].
For the SDS-PAGE protein samples were separated on 10% polyacrylamide gels, topped by a 4% stacking gel using the Mini-Protean 3 apparatus (Bio-Rad, Hercules, CA, USA). Proteins were directly transferred onto nitrocellulose membranes in blotting buffer for 2 h at 4 °C. Thereafter, nitrocellulose membranes were incubated in blocking solution (1% BSA/TBS/0.1% Tween-20,) for 1 h at RT. Membranes were incubated with polyclonal rabbit anti-lysenin antibody (1:1500, Pepta Nova GmbH, Sandhausen, Germany) [2] or mouse monoclonal anti-tubulin (1:1000, Sigma-Aldrich) as a loading control in blocking solution overnight at 4 o C. On the following day, membranes were washed in TBS-T for 30 min and incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:1000) or anti-mouse IgG secondary antibody (1:1000) in TBS-T for 1 h at RT. Followed the washing steps ECL detection reagent (Super Signal West Pico Plus, Thermo Scientific) was used to visualize the membranes. Chemiluminescent signals were detected by ChemiDoc Imaging System and analyzed by ImageLab software (BioRad).

RNA-isolation, cDNA synthesis and real-time PCR
Total RNA was extracted from intact and regenerating tissues using NucleoZOL reagent (Macherey-Nagel GmbH, Düren, Germany) and Potter Grinding Chamber (Proscientific, Oxford, CT, USA) according to the manufacturer's instructions. RNA quantity and quality was ascertained with a NanoDrop spectrophotometer. RNA samples were stored at -80 °C. Prior to cDNA preparation, DNase digestion (Amplification Grade DNase I; Sigma-Aldrich) was performed (25 °C for 15 min, 72 °C for 10 min) in order to achieve greater RNA purity. Next, DNase-digested total RNA was transcribed to cDNA using a High Capacity cDNA Reverse Transcription Kit (Thermo Scientific) based on the manufacturer's protocol. Prepared cDNAs were stored at -20 °C and subsequently used as PCR reaction templates [3].
Gene-specific primers [3,4] were designed by Primer Express Software (Thermo Scientific) (please see Table S1.). Gene expressions were measured by qPCR using a Maxima SYBR Green Master Mix (Thermo Scientific) on an ABI Prism 7500 (Applied Biosystems). The predenaturation step of the amplification profile started at 95 °C and lasted for 10 minutes. This was followed by 40 cycles of denaturation (35 s at 95 °C), hybridization (35 s at 58 °C), and elongation (1 min at 72 °C) stages with ultimately a dissociation step. Quantitative measurements were normalized to RPL17 mRNA level [3]. PCR data were determined from four independent experiments. Tables   Table S1. Lists of primers and GenBank Accession numbers used for qPCR analysis

Target gene
Gene Bank accession #