INTRODUCTION: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. METHODS: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria - Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. RESULTS: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. DISCUSSION: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.

• Klíčová slova
• Bacillus thuringiensis, comparative immune response, gut immunity, hematopoietic organ, insect immunity, insect midgut, lysozyme,
• MeSH
• larva MeSH
• Manduca * genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• muramidasa genetika   metabolismus   MeSH
• přirozená imunita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• muramidasa MeSH

Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.

• Klíčová slova
• PRR, TLR, development, earthworm, gregarine, innate immunity, invertebrate, parasite,
• MeSH
• cystein MeSH
• cytokiny imunologie   MeSH
• embryonální vývoj imunologie   MeSH
• fylogeneze MeSH
• messenger RNA imunologie   MeSH
• nádorové proteiny imunologie   MeSH
• NF-kappa B imunologie   MeSH
• Oligochaeta imunologie   MeSH
• přirozená imunita imunologie   MeSH
• proteiny vázající RNA imunologie   MeSH
• receptory rozpoznávající vzory imunologie   MeSH
• signální transdukce imunologie   MeSH
• toll-like receptory imunologie   MeSH
• Toxoplasma imunologie   MeSH
• upregulace imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• cytokiny MeSH
• messenger RNA MeSH
• nádorové proteiny MeSH
• NF-kappa B MeSH
• proteiny vázající RNA MeSH
• receptory rozpoznávající vzory MeSH
• small inducible cytokine subfamily E, member 1 MeSH Prohlížeč
• toll-like receptory MeSH

Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs), together with polycyclic aromatic hydrocarbons (PAHs), represent highly toxic and persistent organic environmental pollutants, especially due to their capability for bioaccumulation in fatty tissues. To observe the environmentally relevant effect of these compounds on earthworms, two soils naturally contaminated with PCDD/Fs and PAHs were used in our experiments. We focused on the role of CuZn- and Mn-superoxide dismutases. We assembled a full-length sequences of these molecules from Eisenia andrei earthworm and confirmed their activity. We demonstrated the significant reduction of CuZn-SOD on both mRNA and enzyme activity levels and increased levels of reactive oxygen species in earthworms kept in PCDD/F-polluted soil, which corresponds to the observed histopathologies of the earthworm intestinal wall and adjacent chloragogenous tissue. The results show an important role of CuZn-SOD in earthworm tissue damage caused by PCDD/Fs present in soil. We did not detect any significant changes in the mRNA expression or activity of Mn-SOD in these earthworms. In earthworms maintained in PAH-polluted soil the activity of both CuZn-SOD and Mn-SOD significantly increased. No histopathological changes were detected in these worms, however significant decrease of coelomocyte viability was observed. This reduced viability was most likely independent of oxidative stress.

• Klíčová slova
• Dioxins, Earthworm, Histopathology, Polycyclic aromatic hydrocarbons, Reactive oxygen species, Superoxide dismutase,
• MeSH
• látky znečišťující půdu toxicita   MeSH
• Oligochaeta anatomie a histologie   účinky léků   enzymologie   MeSH
• polychlorované dibenzodioxiny toxicita   MeSH
• polychlorované dibenzofurany toxicita   MeSH
• polycyklické aromatické uhlovodíky toxicita   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• superoxid dismutáza 1 genetika   metabolismus   MeSH
• superoxiddismutasa genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• látky znečišťující půdu MeSH
• polychlorované dibenzodioxiny MeSH
• polychlorované dibenzofurany MeSH
• polycyklické aromatické uhlovodíky MeSH
• reaktivní formy kyslíku MeSH
• superoxid dismutáza 1 MeSH
• superoxiddismutasa MeSH

Vermicomposting is a process of degradation of biowaste which involves complex interactions between earthworms and microorganisms. This process lacks a thermophilic stage and thus, the possible presence of pathogens poses a potential health hazard. To assess the contribution of earthworms during the selective reduction of various pathogens, apple pomace substrate was artificially inoculated with Escherichia coli, Salmonella spp., thermotolerant coliform bacteria, and Enterococci. The artificial bacterial load did not influence the weight, reproduction, or intestinal enzymatic activity of the earthworms, but it caused reversible histological changes to the epithelial layer and chloragogen tissue of their intestines. The reduction of pathogenic Enterococci and E. coli from the substrate was accelerated by earthworms (63-fold, 77-fold, and 840-fold for Enterococci and 6-fold, 36-fold, and 7-fold for E. coli inoculated substrates after 2, 4, and 6 weeks, respectively). Moreover, the rapid elimination of Salmonella spp. was supported by the upregulated expression of two pattern recognition receptors which bind lipopolysaccharide, coelomic cytolytic factor, and lipopolysaccharide-binding protein. Further, the microbiomes of the intestine and the composting substrate differed significantly. Graphical abstract.

• Klíčová slova
• Biowaste, Earthworm, Eisenia, Immunity, Microbiome, Pathogen, Pattern recognition receptor, Vermicompost,
• MeSH
• Escherichia coli MeSH
• kompostování metody   MeSH
• Oligochaeta mikrobiologie   fyziologie   MeSH
• půdní mikrobiologie * MeSH
• střevní mikroflóra * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.

• Klíčová slova
• CCF, Coelomocyte, Earthworm, Microorganism, Mucosal immunity,
• MeSH
• Bacillus subtilis imunologie   MeSH
• buněčná imunita MeSH
• Escherichia coli imunologie   MeSH
• gramnegativní bakteriální infekce imunologie   MeSH
• grampozitivní bakteriální infekce imunologie   MeSH
• lektiny genetika   metabolismus   MeSH
• mezoderm imunologie   patologie   MeSH
• mykózy imunologie   MeSH
• Oligochaeta imunologie   MeSH
• receptory rozpoznávající vzory genetika   metabolismus   MeSH
• Saccharomyces imunologie   MeSH
• slizniční imunita MeSH
• střevní sliznice imunologie   mikrobiologie   virologie   MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• coelomic cytolytic factor 1, Eisenia fetida MeSH Prohlížeč
• lektiny MeSH
• receptory rozpoznávající vzory MeSH

LBP/BPIs are pattern recognition receptors that are often present in vertebrates and in invertebrates, and they play a defense role against pathogens. We have identified 1698 bp cDNA sequence from the Eisenia andrei earthworm with predicted amino acid sequence that shares homology with the LBP/BPI family (EaLBP/BPI). Sequence analysis of EaLBP/BPI proved the existence of two conserved domains with the potential ability to bind LPS. The predicted molecular mass of the EaLBP/BPI protein is 53.5 kDa, and its high basicity (pI 9.8) is caused by its high arginine content. Constitutive transcription of the Ealbp/bpi gene was shown in all tested tissues, with the highest level in coelomocytes and seminal vesicles; the lowest level was detected in the intestine. On the contrary, another earthworm LPS-binding molecule CCF (coelomic cytolytic factor) was expressed only in the intestine and coelomocytes. In E. andrei coelomocytes, the transcription of Ealbp/bpi gene was up-regulated in response to bacterial stimulation, reaching a maximum at 8 and 16 h post stimulation with Bacillus subtilis and Escherichia coli, respectively.

• Klíčová slova
• CCF, Earthworm, Eisenia, LBP/BPI, LPS, PRR,
• MeSH
• fylogeneze MeSH
• hybridizace in situ MeSH
• kationické antimikrobiální peptidy genetika   MeSH
• klonování DNA MeSH
• krevní proteiny genetika   MeSH
• molekulární sekvence - údaje MeSH
• Oligochaeta genetika   imunologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bactericidal permeability increasing protein MeSH Prohlížeč
• kationické antimikrobiální peptidy MeSH
• krevní proteiny MeSH

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5'- or 3'-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

• MeSH
• akonitáthydratasa metabolismus   MeSH
• ferritin metabolismus   MeSH
• fylogeneze MeSH
• homeostáza fyziologie   MeSH
• konformace nukleové kyseliny MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• messenger RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• Oligochaeta růst a vývoj   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny regulující obsah železa genetika   metabolismus   MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akonitáthydratasa MeSH
• ferritin MeSH
• messenger RNA MeSH
• proteiny regulující obsah železa MeSH
• železo MeSH

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) belong to the group of persistent organic pollutants, highly toxic environmental pollutants that include hydrophobic compounds with the tendency to bioaccumulate. Earthworms (Eisenia andrei) were exposed to PCDD/Fs-contaminated soil, and changes in their lipophilic structures and the gene expression of their defense molecules were followed. Damage to the intestinal wall and adjacent chloragogenous tissue was observed. Further, the up-regulation of the expression of several genes was detected. On the basis of these results, the mechanism of the impact of PCDD/Fs on earthworms has been proposed. Dioxins that accumulate in the lipophilic structures cause an increase in reactive oxidative species that triggers oxidative stress followed by the gene expression of two molecules that play a role in protection against oxidant toxicity, calreticulin (CRT) and Hsp70. Moreover, the effect of microbial biomass on the expression of coelomic cytolytic factor (CCF), a pattern recognition receptor, was also observed.

• Klíčová slova
• CCF, Calreticulin, Chloragogenous tissue, Eisenia andrei, Hsp70, PCDD/Fs, ROS,
• MeSH
• benzofurany analýza   metabolismus   MeSH
• dioxiny analýza   metabolismus   MeSH
• látky znečišťující půdu analýza   metabolismus   MeSH
• Oligochaeta účinky léků   genetika   fyziologie   MeSH
• půda chemie   MeSH
• regulace genové exprese účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzofurany MeSH
• dibenzo(1,4)dioxin MeSH Prohlížeč
• dibenzofuran MeSH Prohlížeč
• dioxiny MeSH
• látky znečišťující půdu MeSH
• půda MeSH

Toll-like receptors (TLRs) play an important role in defense responses to pathogens in invertebrates. Here we characterize the first TLR isolated from an oligochaete annelid, namely, Eisenia andrei (EaTLR) and show its expression pattern. The full-length EaTLR cDNA consists of 2615 bp encoding a putative protein of 675 amino acids. The predicted amino acid sequence comprises of an extracellular domain containing 31 amino acid signal peptide and seven leucine-rich repeats (LRR), capped with cysteine-rich N- and C-terminal LRRs followed by a transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). TIR domains of twenty individual earthworms were sequenced and the variability suggesting the presence of a high number of TLR genes in the genome of E. andrei was observed. Phylogenetic analysis revealed the highest similarity of EaTLR with polychaete annelid, Capitella teleta and TLRs of mollusks and echinoderms. Finally, the highest constitutive expression of EaTLR was observed in the digestive tract. Gene expression was significantly increased in coelomocytes of E. andrei after the challenge with Gram-positive bacteria.

• Klíčová slova
• EaTLR, Eisenia andrei, Eisenia andrei Toll-like receptor, Innate immunity, Invertebrates, LRR, PAMPs, PRR, TLR, Toll-like receptor, leucine-rich repeat TIR, Toll/IL-1R domain, pathogen-associated molecular patterns, pattern recognition receptor,
• MeSH
• exprese genu MeSH
• fylogeneze MeSH
• klonování DNA metody   MeSH
• molekulární sekvence - údaje MeSH
• Oligochaeta genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• toll-like receptory biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• toll-like receptory MeSH

Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression.

• Klíčová slova
• Digestive enzyme, Earthworm, Eisenia andrei, Gut, Innate immunity, Microbiota,
• MeSH
• Bacillus subtilis imunologie   MeSH
• celulasy metabolismus   MeSH
• Escherichia coli imunologie   MeSH
• hexosaminidasy metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• muramidasa metabolismus   fyziologie   MeSH
• Oligochaeta enzymologie   imunologie   mikrobiologie   MeSH
• přirozená imunita MeSH
• proteasy metabolismus   MeSH
• Saccharomyces cerevisiae imunologie   MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• celulasy MeSH
• hexosaminidasy MeSH
• messenger RNA MeSH
• muramidasa MeSH
• proteasy MeSH