The metabolites of the manumycin family are known to have a broad range of antibiotic functions including antibacterial, anticoccidial, and antifungal activities (1). Manumycin compounds also display a strong activity against farnesyltransferase, IB kinase ␤, interleukin-1␤-converting enzymes, and acetylcholinesterase and are considered as drug candidates to treat cancers, inflammation, and Alzheimer disease (1-4). Asukamycin A1, a manumycin-type metabolite, contains a unique 2-amino-4-hydroxy-5,6-epoxycyclohex-2-enone (mC 7 N) 3 core and two trans-triene polyketide chains, in which the upper one starts with a cyclohexane ring, and the lower one terminates in the five-membered ring of a 2-amino-3-hydroxycyclopent-2-enone (C 5 N) moiety (Fig.

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Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.

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 ( Ea TLR), 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 mcc Ea TLR 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 mcc Ea TLR 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 mcc Ea TLR mRNA, presumably, earthworm sperm cells have a certain level of mcc Ea TLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mcc Ea TLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mcc Ea TLR 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, mcc Ea TLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.