Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei.

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

Peptidoglycan recognition proteins from the earthworm, Eisenia andrei: Differential inducibility and tissue-specific expression.

Several pattern recognition receptors (PRRs) involved in innate immunity have been identified and characterized in earthworms. Peptidoglycan recognition proteins (PGRPs) are highly conserved PRRs that activate effector pathways such as prophenoloxidase cascade and Toll-like receptor pathway. In addition, PGRPs function as an enzyme, N-acetylmuramoyl-l-alanine amidase (NAMLAA), to directly hydrolyze peptidoglycan. We identified four full-length complementary DNA (cDNA) sequences, Ean-PGRP1/2/3/4, in Eisenia andrei, an earthworm. Sequence and phylogenetic analyses indicate that earthworm PGRP orthologs resemble short PGRP member proteins. The subcellular localizations of four Ean-PGRPs lacking the transmembrane domain are predicted to be extracellular or cytoplasmic. All Ean-PGRPs contain a highly conserved PGRP domain with a conserved Zn2+ binding site including a tyrosine residue essential for active amidase activity. Three highly conserved amino-acid residues (His, Trp, and Thr) necessary for amidase activity are also present, indicating that the Ean-PGRPs can be predicted to have amidase activity. Furthermore, we demonstrate that the Ean-PGRP genes are differentially induced by certain bacterial species, suggesting that the innate immune system of earthworms is likely to be somewhat specific rather than entirely non-specific. Tissue expression patterns indicate that Ean-PGRP mRNAs are primarily expressed in the immune-competent tissues and that their expression is tissue-specific according to Ean-PGRP types, particularly for Ean-PGRP1.

Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity.

BACKGROUND: Chitinase is a multi-functional enzyme that catalyzes the hydrolysis of ß-1,4-linkages between N-acetylglucosamines (GlcNAc) in chitin. Recent studies imply that earthworm chitinase is implicated in self-defense immunity against chitin-containing pathogens. However, a direct relationship of earthworm chitinase with innate immunity has not yet been established. OBJECTIVE: In this study, earthworm (Eisenia andrei) chitinase expression was examined following bacterial challenge by Bacillus subtilis. METHODS: RNA sequencing (RNA-seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to quantitatively evaluate mRNA expression changes in response to bacterial stimulation. RESULTS: Multiple chitinase-related mRNAs were found to be upregulated, among which EaChi3, EaChi4, and EaChi2 were upregulated by approximately eightfold, eightfold, and 2.5-fold, respectively. This strongly suggested that earthworm chitinases may act as inducible humoral effectors in earthworm innate immunity. The primary structures of all three chitinases contained an N-terminal glycol_18 domain with two chitin-binding and chitin-catalyzing domains, and a C-terminal proline, glycine, serine, threonine (PGST)-rich domain. In addition, EaChi2 had a chitin-binding peritrophin-A domain at the end of the C-terminus with 5 cysteine residues possibly contributing two intradomain disulfide bonds. Multiple sequence alignment of the catalytic domain centers of glycol_18 domain displayed highly conserved chitin-binding and chitin-catalyzing domains in which three essential amino acid residues (D, D, E) for catalyzing activity are well conserved except EaChi4. The critical glutamic acid (E) residue was substituted for glutamine (Q) in EaChi4 indicating that it is devoid of catalytic activity. CONCLUSIONS: To our knowledge, this is the first report providing direct evidence that multiple earthworm chitinases are bacteria-responsive, strongly suggesting that earthworm chitinases are inducible humoral effectors in earthworm innate immunity. In addition, our results possibly suggest that earthworm EaChi4 may function as a pattern recognition molecule modulating the downstream immune pathway.

Evaluation of pig behavior changes related to temperature, relative humidity, volatile organic compounds, and illuminance.

The objective of this study was evaluation of pig behavior changes related to temperature, relative humidity, volatile organic compounds (VOCs), and illuminance. A total of 24 growing pigs ([Yorkshire × Landrace] × Duroc) were used in the experiment. A sensor was installed at a height of 0.5 m in the center of the pig house. In experiment 1, temperature was changed every four days to 18°C (T1), 22°C (T2), 26°C (T3), and then 30°C (T4). In experiment 2, relative humidity was adjusted to 45% (low humidity [LH]), 60% (middle humidity [MH]), and then 75% (high humidity [HH]) for four days. In experiment 3, after cleaning the pig house just before experiment, only minimal ventilation was provided. VOCs and pig behaviors were observed for 7 days without cleaning the pig house. In experiment 4, three light bulbs of 40 W (470 lumens / 45 lx; low illuminance [LI]), 75 W (1,055 lumens / 103 lx; middle illuminance [MI]), and 100 W (1,521 lumens / 146 lx; high illuminance [HI]) were used for four days each. Pig behavior analysis was performed for following criteria : Feed intake, Standing, Lying, Sitting, Drink water, Rooting, Posture transition (lying-standing), Posture transition (standing-lying), Wallowing, and Biting. In experiment 1, feed intake time was lower (p < 0.05) for the T3 than other treatment groups. Standing time was highest (p < 0.05) for the T1 and lowest (p < 0.05) for the T3. Lying time was shorter (p < 0.05) in T1 and T2 compared to T3 and T4. Drinking frequency was higher (p < 0.05) for the T4 than other treatment groups. In experiment 2, the frequency of rooting and wallowing increased (p < 0.05) with increasing humidity. LH showed the lowest (p < 0.05) rooting frequency and HH showed the highest (p < 0.05) rooting frequency. In experiment 3, VOCs concentration did not (p > 0.05) change pig behavior. In experiment 4, lying time was the longest (p < 0.05) at LI and shortest (p < 0.05) at HI. Therefore, pig behavior is heavily influenced by the environment, especially temperature and humidity. However, correlation between pig behavior to VOCs and illuminance seems to be needed more research.

Identification and Spatiotemporal Expression of Adenosine Deaminases Acting on RNA (ADAR) during Earthworm Regeneration: Its Possible Implication in Muscle Redifferentiation.

Adenosine deaminases acting on RNA (ADAR) catalyze the hydrolytic deamination of adenosine (A) to produce inosine (I) in double-stranded RNA substrates. A-to-I RNA editing has increasingly broad physiological significance in development, carcinogenesis, and environmental adaptation. Perionyx excavatus is an earthworm with potent regenerative potential; it can regenerate the head and tail and is an advantageous model system to investigate the molecular mechanisms of regeneration. During RNA sequencing analysis of P. excavatus regenerates, we identified an ADAR homolog (Pex-ADAR), which led us to examine its spatial and temporal expression to comprehend how Pex-ADAR is linked to regeneration. At first, in domain analysis, we discovered that Pex-ADAR only has one double-stranded RNA-binding domain (dsRBD) and a deaminase domain without a Z-DNA-binding domain (ZBD). In addition, a comparison of the core deaminase domains of Pex-ADAR with those of other ADAR family members indicated that Pex-ADAR comprises the conserved three active-site motifs and a glutamate residue for catalytic activity. Pex-ADAR also shares 11 conserved residues, a characteristic of ADAR1, supporting that Pex-ADAR is a member of ADAR1 class. Its temporal expression was remarkably low in the early stages of regeneration before suddenly increasing at 10 days post amputation (dpa) when diverse cell types and tissues were being regenerated. In situ hybridization of Pex-ADAR messenger RNA (mRNA) indicated that the main expression was observed in regenerating muscle layers and related connective tissues. Taken together, the present results demonstrate that an RNA-editing enzyme, Pex-ADAR, is implicated in muscle redifferentiation during earthworm regeneration.

Effect of replacing corn with soy hulls on nutrient digestibility of growing pigs.

The objective of this study was to determine the effect of replacing corn with soy hulls on nutrient digestibility of growing pigs. Three experimental diets were tested using a 3 × 3 Latin square design using three barrows per group (Landrace × Yorkshire × Duroc, average initial body weight of 36.9 ± 4.0 kg) in individual metabolic cages. The dietary treatments were the control (CON) basal diet (corn-soybean meal based diet), whereas in the test diets, soy hulls were included at 5% (H1), 10% (H2), gradually replacing corn. The daily feed allowance was adjusted to 2.7 times the maintenance requirement for digestible energy (DE) (2.7 × 110 kcal of DE/kg BW0.75). Intake of crude fiber (CF) was different among treatments (p < 0.05). Pigs fed with H2 diet excreted higher concentrations of dry matter (DM), and CF than pigs fed with H1 diet and basal diet (p < 0.05). Pigs fed with H1 and H2 diets excreted higher concentrations of crude protein (CP) than pigs fed with CON diet (p < 0.05). The apparent total tract digestibility (ATTD) of CF from H1 and H2 diets were higher than CON diet (p < 0.05). Pigs fed with H2 diets were significantly lower (p < 0.05) the ATTD of CP than the pigs fed with CON and H1 diets. The current data suggest that 10% inclusion soy hulls can slightly decrease CP digestibility. However, soy hulls considerably increased CF digestibility.It is concluded that 5% soy hulls may be a usable alternative to corn in growing pig diets.