Antimicrobial defense mechanisms in the horseshoe crab,Limulus polyphemus: Effect of sodium chloride on bactericidal activity

Bactericidal activity of heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) was found to be inhibited by low molar (10 to 100 mm) concentrations of the monovalent cations Na⁺ and K⁺. The protective effect of Na⁺ was shown to be species dependent and inversely proportional to the sensitivity of the test microorganism as determined by bactericidal titer. The effect of Na⁺ (100 mm) on bactericidal activity in whole (unheated) LAL, when assayed against a sensitive species (Escherichia coli), was negligible and in direct contrast to data obtained when using 60°C extracts of LAL. Increasing the concentration of Na⁺ to reflect levels in L. polyphemus blood (400 to 500 mm) afforded only minimal protection for sensitive bacterial species. These data suggested that whole LAL may contain a heat-labile substance (protein?) capable of binding Na⁺, obviating the inhibitory effect of Na⁺ on bactericidal activity. Further evidence for the existence of this cation-binding substance was obtained when 55°C extracts of LAL were found refractory to Na⁺ concentrations which totally abolished bactericidal activity in 60°C extracts. The mechanism of Na⁺ inhibition of bactericidal activity and the role of the cation-binding substance in L. polyphemus host defense against bacterial invasion and dissemination is discussed.     

Cellular biomarkers to elucidate global warming effects on Antarctic sea urchin Sterechinus neumayeri

Global warming is a reality and its effects have been widely studied. However, the consequences for marine invertebrates remain poorly understood. Thus, the present study proposed to evaluate the effect of elevated temperature on the innate immune system of Antarctic sea urchin Sterechinus neumayeri. Sea urchins were collected nearby Brazilian Antarctic Station ??Comandante Ferraz?? and exposed to 0 (control), 2 and 4°C for periods of 48?h, 2, 7 and 14?days. After the experimental periods, coelomic fluid was collected in order to perform the following analyses: coelomocytes differential counting, phagocytic response, adhesion and spreading coelomocytes assay, intranuclear iron crystalloid and ultra structural analysis of coelomocytes. The red sphere cell was considered a biomarker for heat stress, as they increased in acute stress. Besides that, a significant increase in phagocytic indexes was observed at 2°C coinciding with a significant increase of intranuclear iron crystalloid at the same temperature and same time period. Furthermore, significant alterations in cell adhesion and spreading were observed in elevated temperatures. The ultra structural analysis of coelomocytes showed no significant difference across treatments. This was the first time that innate immune response alterations were observed in response to elevated temperature in a Polar echinoid.     

Antibacterial activities of the methanol extracts of Canarium schweinfurthii and four other Cameroonian dietary plants against multi-drug resistant Gram-negative bacteria

Bacterial infections are among the major cause of morbidity and mortality worldwide. The present study was designed to evaluate the in vitro antibacterial activities of the methanol extracts of five Cameroonian edible plants namely Colocasia esculenta, Triumfetta pentandra, Hibiscus esculentus, Canarium schweinfurthii and Annona muricata against a panel of 19 multidrug resistant Gram-negative bacterial strains. The liquid broth microdilution was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extracts. The preliminary phytochemical screening of the extracts was conducted according to the standard phytochemical methods. Results showed that all extracts contained compounds belonging to the classes of polyphenols, triterpenes and steroids, other classes of chemicals being selectively distributed. Canarium schweinfurthii extract showed the best activity with MIC values ranging from 64 to 1024 μg/mL against 89.5% of the 19 tested bacteria strains. MIC values below or equal to 1024 μg/mL were also recorded with Triumfetta pentandra, Annona muricata, Colocasia esculenta and Hibiscus esculentus extracts respectively against 15/19 (78.9%), 11/19 (57.9%), 10/19 (52.6%) and 10/19 (52.6%) tested bacteria. Extract from C. schweinfurthii displayed the lowest MIC value (64 μg/mL) against Escherichia coli AG100A_Tet. Finally, the results of this work provide baseline information for the use of C. esculenta, T. pentandra, H. esculentus, C. schweinfurthii and A. muricata in the treatment of bacterial infections including multidrug resistant phenotypes.     

Citric acid production from imumu Cyperus esculentus and Maize Zea mays

The production of citric acid from imumu Cyperus esculentus and maize Zea mays was carried out using Aspergillus niger. Hydrolysis of the substrates at 97°C produced a higher concentration of reducing sugars than at 40°C. The concentrations of citric acid were higher in unagitated and defatted cultures compared to agitated and undefatted cultures respectively.     

The role of chemical signals in the spawning induction of polychaete worms and other marine invertebrates

Extracts of gonad, body fluid and spawning water (into which gametes have been released by the process of spawning) were tested for the presence of spawning-inducing activity (SIA) on both sexes of the polychaetes Nereis succinea (Frey and Leuckhart) and Platynereis dumerilii (Audouin and Milne-Edwards). Gonadal and body fluid extracts from Asterias rubens (L.) and Echinus esculentus (L.) (echinoderms), Arenicola marina (L.) (polychaete), Clupea harengus (L.) (teleost) and body fluid and spawning water of Nereis virens (Sars) (polychaete) all exhibit SIA on male N. succinea. C. harengus extract from male gonads also had SIA on male P. dumerilii. Phytoplankton monoculture extracts were also tested. The cryptomonad (Rhodomonas baltica) (Karsten) had SIA on male P. dumerilii, and the diatom Phaeodactylum tricornutum (Bohlin) had some activity on Mytilus edulis (L.). The active fraction of R. baltica is chromatographically similar to uric acid (the natural sperm-release pheromone produced by females of P. dumerilii) [Nature 382 (1996) 214], but has a different retention time and UV absorbance spectra on reverse phase HPLC. Conspecific extracts of spawning water and gonadal material did not have SIA on the same and opposite sex in A. marina and N. virens. The role of chemical signals in marine invertebrates is discussed in relation to their mode of spawning.     

Biochemical composition, biological activities and toxicological effects of two non-nodularin producing strains of Nodularia spumigena Mertens in Jürgens

We tested two non-nodularin-producing strains of the cyanobacterium Nodularia spumigena, isolated from a marine (Kachelotplate) and a brackish water (Banter Sea, Wilhelmshaven) habitat in Lower Saxony, Germany, for allelochemical production (e.g. alkaloids, flavonoids) and allelopathic activities (e.g. algicidal, anti-microbial). The growth experiments showed for the marine strain the highest cell density at 10 and 20 °C for the brackish water isolate (80 μmol ?photons m^?2?s^?1). Phytochemical screening of the biomass extracts gave positive results for alkaloids, flavonoids, sterols and terpenoids in some of the tested assays. Most of these compounds were not present in supernatant extracts. Besides proalgal and anti-cyanobacterial properties of the high temperature treated marine strain, the supernatant extracts showed profungal and antibacterial activities in the 20 °C treated assays. In both, supernatant and biomass extracts, significant anti-oxidative activities were observed in the high-irradiance-treated marine and brackish water isolates. The highest toxicity was observed at the 5 and 20 °C brackish water isolates as well as 5 °C treated marine strain. With regard to fatty acid composition, both strains showed high levels of polyunsaturated fatty acids (PUFAs) and saturated fatty acids, with values of 36–54 % and 11–29 % of total fatty acids, respectively, whereas the levels of monounsaturated fatty acids were in general lower (8–16 %). Among PUFAs, linoleic (C18:2), α-linoleic (C18:3), γ-linoleic (C18:3) and arachidonic acid (C20:4) accounted 36.2 % of the total polyunsaturated fatty acids in the brackish water strain, while in the marine isolate, it was only 10.6 %.     

A comparative study of the bactericidal activity of horseshoe crab (Limulus polyphemus) hemolymph and vertebrate serum

This paper describes a partially heat-labile, naturally occurring bactericidal factor in cell-free hemolymph preparations obtained from Limulus polyphemus. This bactericidal activity has been shown to be directed against two Gram-negative bacteria, Escherichia coli and Klebsiella pneumoniae, whereas it had no effect on the Gram-positive bacteria tested, Micrococcus lysodeikticus and Staphylococcus aureus. Maximal bactericidal activity of this factor was observed at 30°C and pH 6.0. Since complement and antibody are required for antimicrobial activity in vertebrate sera, the activity of this factor in the presence of various complement inhibitors was assayed. The bactericidal activity of Limulus hemolymph is abolished by treatment with endotoxin; however, other anticomplementary substances were without effect. Limulus amebocyte lysate is known to contain protein which may be precipitated by endotoxin; it is possible that the reduction of bactericidal activity produced by endotoxin treatment may be caused by the denaturation of a bactericidal protein moiety produced by the hemocytes.     

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.     

The determination of activity of the enzyme Rubisco in cell extracts of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short‐term thermal stress of the alga

The dinoflagellate alga Symbiodinium sp., living in symbiosis with corals, clams and other invertebrates, is a primary producer in coral reefs and other marine ecosystems. The function of the carbon‐fixing enzyme ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in dinoflagellates is difficult to study because its activity is rapidly lost after extraction from the cell. We report procedures for the extraction of Rubisco from Symbiodinium cells and for stable storage. We describe a continuous assay for Rubisco activity in these crude cell extracts using the Mn²⁺ chemiluminescence of Rubisco oxygenase. Chemiluminescence time courses exhibited initial transients resembling bacterial Form II Rubisco, followed by several minutes of linearly decreasing activity. The initial activity was determined from extrapolation of this linear section of the time course. The activity of fast‐frozen cell extracts was stable at ?80 °C and, after thawing and storage on ice, remained stable for up to 1 h before declining non‐linearly. Crude cell extracts bound [¹⁴C] 2‐carboxy‐D‐arabitinol 1,5‐bisphosphate to a high molecular mass fraction separable by gel filtration chromatography. After pre‐treatment of Symbiodinium cell cultures in darkness at temperatures above 30 °C, the extracted Rubisco activities decreased, with almost complete loss of activity above 36 °C. The implications for the sensitivity to elevated temperature of Symbiodinium photosynthesis are assessed.     

Neutrophil migration in canines: In vivo comparison of granulocyte function following 24-hour storage at 6 or 20 °C of leukocyte concentrates or of granulocytes purified by counterflow centrifugation-elutriation

The use of granulocyte-rich concentrates from leukapheresis purified by counterflow centrifugation—elutriation to obtain pure granulocytes for transfusion studies in cyclo-phosphamide-induced neutropenic animal models is reported. Our data for granulocyterich leukapheresis concentrates indicate that room temperature (20 °C) appears to be preferred to 6 °C for short-term granulocyte storage. The data also indicate that although the granulocytes isolated by counterflow centrifugation—elutration may retain in vitro functions of chemotaxis, phagocytosis, and bactericidal activity, the in vivo function of migration into skin chambers for isolated granulocytes is seriously impaired after storage for 18 to 24 hr at both 6 and 20 °C. This loss of in vivo function of stored granulocytes occurs in isolated granulocytes obtained by both counterflow centrifugation-elutriation and dextran sedimentation, and it is not observed in the leukocyte concentrates held at 20 °C. The results of these studies are fourfold. First, freshly isolated granulocytes display no apparent loss of either in vivo or in vitro function. Second, granulocytes isolated by counterflow centrifugation-elutriation or dextran sedimentation and stored at 6 or 20 °C are severely impaired in terms of their in vivo chemotactic function but display no loss of in vitro efficacy. Third, 20 °C storage of granulocyte-rich leukapheresis concentrates for 18 to 24 hr is superior to 6 °C storage. Fourth, in vitro analysis may be limited in its ability to indicate in vivo function as a measure of success in granulocyte preservation studies.     

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).     

Inactivation of endotoxin by Limulus amoebocyte lysate.

The inactivation of bacterial endotoxin by aqueous extracts (Limulus amoebocyte lysate) of the circulating blood cells (amoebocytes) of the horseshoe crab, Limulus polyphemus, is described. Active extracts were obtained by heating Limulus amoebocyte lystate (LAL) to 60°C for 20 min to denature the clotting enzyme, rendering the LAL incapable of gel formation in the presence of endotoxin. Endotoxin inactivation was assayed using the Limulus amoebocyte lysate test and by rabbit bioassay. Inactivation of endotoxin with heated extracts of LAL was suggestive of enzymatic mediation, as indicated by dependence on time, temperature, pH, and the kinetics of inactivation. Endotoxin inactivation occurred over a broad pH range, 4.5–8.5, with the optimum at a pH of 6.1. Temperature optima were between 37° and 50°C, with observed activity between 0° and 65°C. Ionized calcium was inhibitory to endotoxin inactivation with heated extracts of LAL, with partial inhibition at 0.001 m calcium and complete inhibition at 0.02 m calcium. Other divalent cations (Mg, Ba, Mn, and Cu) were also found to inhibit the inactivation of endotoxin. Similarities between the endotoxin-inactivating system of L. polyphemus and those described to be present in mammalian and lower vertebrate sera are discussed.     

Plant growth promoting activity of seaweed liquid extracts produced from Macrocystis pyrifera under different pH and temperature conditions

Most commercial algal extracts are produced from brown algae by alkaline hydrolysis; however, little scientific information has been published regarding the details of the production process. In this research, we have investigated the effect of pH (pH 8–12) and temperature (40, 60, and 80 °C) on liquid extract production from the brown alga Macrocystis pyrifera. Production conditions influenced the physicochemical characteristics of the final product as the extract viscosity increased with increasing pH and temperature to a maximum which occurred at pH 10 and 80 °C. This suggests that at higher pH conditions, alginate and other polysaccharides were extracted. All the extracts obtained promoted growth of tomato plants (Solanum lycopersicum) and adventitious root formation in the mung bean cutting bioassay (Vigna radiata), as the pH process was increased during the production of the liquid extracts. The highest auxin-type activity was obtained with the extract produced at pH 11 and 80 °C, while the fastest tomato seedling growth was achieved with the extract produced at pH 12 and 80 °C.     

Cloning,expression and characterization of a novel cold-active and halophilic xylanase from Zunongwangia profunda

A new xylanase gene (xynA) from the marine microorganism Zunongwangia profunda was identified to encode 374 amino acid residues. Its product (XynA) showed the highest identity (42.78 %) with a xylanase from Bacillus sp. SN5 among the characterized xylanases. XynA exhibited the highest activity at pH 6.5 and 30 °C, retaining 23 and 38 % of the optimal activity at 0 and 5 °C, respectively. XynA was not only cold active, but also halophilic, and both its activity and thermostability could be significantly increased by NaCl, showing the highest activity (180 % of the activity) at 3 M NaCl and retaining nearly 100 % activity at 5 M NaCl, compared to the absence of NaCl. In the presence of 3 M NaCl, the k _cat/K _m value of XynA exhibited a 3.41-fold increase for beechwood xylan compared to no added NaCl, and the residual activity of XynA increased from 23 % (no added NaCl) to 58 % after 1 h incubation at 45 °C. This may be the first report concerning a cold-adapted xylanase from a non-halophilic species that displays the highest activity at a NaCl concentration range from 3 to 5 M. The features of cold activity and salt tolerance suggest the potential application of XynA in the food industry and bioethanol production from marine seaweeds.     

Antimicrobial Resistance of the Coral Pathogen Vibrio coralliilyticus and Caribbean Sister Phylotypes Isolated from a Diseased Octocoral

Vibrio coralliilyticus is a global marine pathogen that has been found to cause disease in several marine organisms, including corals. This study is the first report of the isolation of V. coralliilyticus from a diseased Caribbean octocoral, Pseudopterogorgia americana. Five sister phylotypes were positively identified using 16S rRNA gene sequencing, recA probes specific for V. coralliilyticus, and rep-PCR fingerprinting. The antimicrobial resistance was compared between pathogenic strains of V. coralliilyticus and the Caribbean strains. First, the antimicrobial resistance of V. coralliilyticus-type strain ATCC BAA-450 was determined using an agar-overlay antimicrobial bioassay at 24°C and 27°C, temperatures which are relevant to its known temperature-dependent virulence. From 108 distinct bacteria isolated from P. americana, 12 inhibited the V. coralliilyticus-type strain at 24°C and five at 27°C. Next, the phenotypic comparison of two Caribbean phylotypes and three V. coralliilyticus reference strains against a subset of 30 bacteria demonstrated a similar resistance trend. At both temperatures, the reference strains were inhibited by three bacteria isolates, while the Caribbean strains were inhibited by four to nine bacteria. Additionally, V. coralliilyticus-type strain ATCC BAA-450 and one of the Caribbean strains were inhibited by a higher number of bacteria at 24°C compared with 27°C. Together, these results highlight that V. coralliilyticus strains have antimicrobial resistance to the majority of coral-associated bacteria tested, which may be temperature-dependent in some strains. Furthermore, all V. coralliilyticus strains tested showed multi-drug resistance to a range of 11–16 (out of 26) commercial antibiotics. This study establishes V. coralliilyticus in association with a Caribbean octocoral and demonstrates its resistance to the antimicrobial activity of coral-associated bacteria and to commercial antibiotics.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

Antimicrobial activity of marine organisms collected off the coast of South East India

In vitro antimicrobial screening of nine marine sponges (Porifera) and two seaweeds, collected from south east coast of India, against selected clinical isolates of bacteria and fungi was conducted in this study. Methanolic extracts of all the marine organisms demonstrated activity against one or more of microbes tested. Sigmadocia carnosa was the most active exhibiting a broad spectrum antimicrobial activity against each of the microbe tested with the exception of Fusarium species. Contrary to this, the genus Echinogorgia did not show any detectable bactericidal activity but, Echninogorgia reticulata was weakly fungicidal against Rhodotorula species and E. compecta against Fusarium and Nocardia species. Considerable antibacterial activity was exhibited by Haliclona cribricutis and Chrotella australiensis against Klebsiella species and Vibrio cholerae, respectively. Petrocia testudinaria showed equally good activity against the bacterium V. chlorae and the fungus Cryptococcus neoformans. The sponges Callyspongia fibrosa, Ircinia species and the seaweed Stoecheospermum margilatum are totally inactive against fungi. The extracts showing good antimicrobial activity are undergoing further analysis to identify the active constituents.     

Bactericidal activity of hemolymph from the horseshoe crab, Limulus polyphemus

Bactericidal activity was found in Limulus serum, with great individual variation in titers toward different bacteria and also among individual horseshoe crabs toward the same bacterial species. These titers varied between monthly determinations of activity. There were crabs with zero activity toward each bacterial species tested. Although environmental factors are likely influences on the bactericidal activity of Limulus serum, the marked variability within similarly treated groups indicates large individual differences in the horseshoe crab population. The highest titers were recorded against those Gram-negative bacilli found normally in the environment. Lower titers were found against those species found normally in warmblooded animals and present in water as contaminants. The serum bactericidal factor is probably released from the circulating amoebocytes during clotting since there was no activity in the “plasma” portion of the blood. Exposure to heat (56°C, 30 min) destroyed the bactericidal activity.     

Cell-free system derived from heat-shocked Escherichia coli: Synthesis of enzyme protein possessing higher specific activity

heat-shocked S30 extract (HS-S30 extract) was prepared from cells of Escherichia coli strain Q13 exposed to elevated temperatures (from 37°C to 42°C) for 30 min. In a cell-free system with HS-S30 extract, the synthesized CAT protein had higher specific activity than that synthesized by a cell-free system with S30 extract prepared from Q13 cells incubated at 37°C. SDS-PAGE analysis showed that the heat-shock proteins, GroEL and DnaK, which are known to be molecular chaperones, were significantly increased in the HS-S30 extract. The addition of GroEL or DnaK to the S30 extract system increased the specific activity of the synthesized CAT protein. Heat-shock induction thus offers an effective method of modifying E. coli cell extracts.     

Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei

Due to the specific habitat conditions in which they live, earthworms are constantly exposed to pathogens. Consequently, they have evolved various immuno-defense mechanisms, including cellular (coelomocytes) and humoral responses, which may help to eliminate deleterious micro-organisms but also repair and/or protect host cells and tissues. Similar to mammalian phagocytes, coelomocytes can kill ingested pathogens with reactive oxygen species (ROS) and nitric oxide. In the present work, we studied the effects of the dermal exposure of Eisenia andrei earthworms to different immuno-stimulants: phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) or concanavalin A (ConA). After 3 days of treatment with all immuno-stimulants, decreased numbers and changed composition of the coelomocytes were observed. The immuno-stimulants also induced numerous changes in bactericidal activity, including ROS production. Furthermore, all stimulants increased cell proliferation while only LPS-treatment significantly elevated apoptosis of coelomocytes. These results demonstrate that in vivo treatment of earthworms with immuno-stimulants induces various changes in their coelomocyte response.