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Distribution of 20 enzymes in the midgut region of the earthworm, Lumbricus terrestris L., with particular emphasis on the physiological role of the chloragog tissue
Abstract
1.1. Lumbricus terrestris midgut was separated into peripheral chloragocytes, peripheral intestinal epithelium and typhlosole, and the distributions of 20 enzymes were assayed.2.2. The peripheral chloragog tissue contained ⩽4% of the midgut dehydrogenase activities, but about 44% or more of the total catalase, Superoxide dismutase, β-d-glucuronidase, alkaline phosphatase, esterase, (δ-aminolevulinate dehydratase and porphyrin synthetase activities.3.3. The midgut contained about 70% or more of the total midbody isocitrate dehydrogenase, glutaminate dehydrogenase and NADH- and NADPH-diaphorase activities.4.4. Most of the intermediary metabolism, including amino acid deamination, probably takes place in the midgut epithelium, which is also the site of the mixed function oxidase system.5.5. The chloragog tissue probably synthesizes the blood hemoglobin and is responsible for the destruction of Superoxide and hydrogen peroxide from the interaction between blood hemoglobin and molecular oxygen.6.6. The high alkaline phosphatase activity in ehloragog tissue may reflect transmembrane transport activity or may be involved in glycogen break-down.7.7. Neither xanthine oxidase nor uric acid, xanthine or hypoxanthine were present in detectable amounts.
... In E. fetida, studies on lysosomal biomarkers in the digestive tract are almost lacking albeit there exists evidence of changes in AcP activity in chloragocytes after exposure to metals (Cancio et al., 1995). About 10% of the gut AcP resides in chloragocytes, both in lysosomes and a small proportion of the chloragosomes (Prentø, 1987;Cancio et al., 1995). β-GUS and esterase activities have also been demonstrated in chloragosomes (Varute and More, 1973). ...
... Catalase is ubiquitous in archaea, prokaryotes, and eukaryotes (Xiong et al., 2013). In earthworms, CAT activity is partitioned between the chloragocyte's cytosol (60-70%) and the gut epithelium peroxisomes (30-40%) (Prentø, 1987). In the absence of environmental insult, one main function of the chloragocyte CAT is scavenging for H 2 O 2 arising from the interaction between blood heme-protein and oxygen (Prentø, 1987) but on exposure to pollutants CAT activity is enhanced to protect against augmented ROS levels (Xiong et al., 2013). ...
... In earthworms, CAT activity is partitioned between the chloragocyte's cytosol (60-70%) and the gut epithelium peroxisomes (30-40%) (Prentø, 1987). In the absence of environmental insult, one main function of the chloragocyte CAT is scavenging for H 2 O 2 arising from the interaction between blood heme-protein and oxygen (Prentø, 1987) but on exposure to pollutants CAT activity is enhanced to protect against augmented ROS levels (Xiong et al., 2013). ...
... Carboxylesterases have been described in earthworms (Haites et al., 1972; Øien and Stenersen, 1984). The intestinal tract of Lumbricus terrestris presents a wide variety of enzyme; among them, CbE activity is predominant in the peripherical chloragog tissue (Prento, 1987). Recently, Hernandez and Wheelock (2009) reported up to twelve CbE isozymes in the gastrointestinal tract, body wall muscle and reproductive tissues of L. terrestris, being particularly abundant in the crop, gizzard and foregut. ...
... Similarly, the digestive secretion of the spider Tegenaria atrica showed six CbE isozymes, which could have also lipolytic activity (Mommsen, 1978). A study by Prento (1987) revealed up to twenty differentTable 1 Kinetic parameters a for hydrolysis of α-naphthyl acetate (α-NA), 4-nitrophenyl acetate (4-NPA) and 4-nitrophenyl valerate (4-NPV) in the tissue homogenates and soil extracts from crop/gizzard and foregut of Lumbricus terrestris. Menten equation or transformation to linearity using the double-reciprocal Lineaweaver–Burk plot in the case of 4-NPV. ...
... In that study, esterase activity using α-NA was particularly abundant in the chloragocyte fraction of the midgut, and it was suggested to be involved in the lipid metabolism. Our results are in line with the previous findings by Prento (1987), and suggest an intestinal secretion of esterases in L. terrestris. However, caution must be taken into account because symbiotic microorganisms could be involved in the presence of the high CbE activity found in the earthworm gut content. ...
Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (alpha-naphthyl acetate [alpha-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (alpha-NA-CbE=8.43+/-2.76U mg(-1) protein and 4-NPA-CbE=5.98+/-2.11U mg(-1) protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K(m) and V(max) values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC(50)) was comparable between tissue (IC(50)s range=4.01-9.67nM dichlorvos and 8480-6880nM paraoxon) and soil (IC(50)s range=6.01-11.5nM dichlorvos and 8400-7260nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes.
... Despite recognition of its potential for the evaluation of sublethal effects of soil pollutants (Venables et al 1992), Hsp induction has been studied only by immunochemistry and in the earthworm species Lumbricus rubellus, either after caudal amputation and parasitic infection (Mariñ o and Morgan 1998) or after exposure to metalliferous soils (Mariñ o et al 1999). In the present study, the species Lumbricus terrestris was chosen because its widespread distribution allows in-thefield assessments and its larger size permits given target tissues or cells to be isolated easily (eg, Ireland 1978; Morgan 1981; Prentø 1987; Diogène et al 1997; Affar et al 1998). Secondly, this particular earthworm species can be readily maintained under laboratory conditions for in vitro and in vivo experiments (eg Morgan 1981; Edwards and Coulson 1992; Li et al 1994; Walsh et al 1995; Diogène et al 1997). ...
... Secondly, this particular earthworm species can be readily maintained under laboratory conditions for in vitro and in vivo experiments (eg Morgan 1981; Edwards and Coulson 1992; Li et al 1994; Walsh et al 1995; Diogène et al 1997). The midgut/intestinal tissues were the main targets selected in the present study since they are key tissues for the assimilation of soil elements (Prentø 1987). Earthworms are also known to be selective in their ingestion of materials (eg, Morgan and Morgan 1992). ...
... Earthworms are also known to be selective in their ingestion of materials (eg, Morgan and Morgan 1992). Moreover, soil pollutants may cause pathophysiological effects on the midgut (eg, Yongcan et al 1998), and many detoxification mechanisms have been associated with midgut tissues (Ireland 1978; Stenersen 1984; Prentø 1987; Morgan et al 1993; Prentø 1994). Therefore, using the Western blot technique, the aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of L. terrestris to various soil pollutants, (2) to assess specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific to a given pollutant or type of pollutant. ...
Induction of heat shock proteins (Hsps) is often associated with a cellular response to a harmful stress or to adverse life conditions. The main aims of the present study were (1) to assess if stress-induced Hsp70 could be used to monitor exposure of the earthworm species Lumbricus terrestris to various soil pollutants, (2) to assess the specificity of pollutants in their tissue targeting and in Hsp70 induction, and (3) to evaluate if dose-response relationships could be established and if the stress-response observed was specific. The midgut/intestinal tissues of L. terrestris are shown to express an inducible member of the Hsp70 family after heat shock treatment in vitro and exposures to different soil toxicants in vivo (re: artificial soil). Short-term (24-72 hours) and long-term (14-16 days) exposures to the chemical standards chloroacetamide and pentachlorophenol and to heavy metals (Pb++, Cd++, Cu++, and Hg++) also affected the earthworms, and Hsp70 was induced in their midgut/intestinal tissues. After a 3-day exposure to heavy metals, the level of Hsp70 induction in the midgut/intestinal tissues appears to correlate well with the reported in vivo and in vitro toxicity data. Comparatively, in proximal and midbody wall muscle tissues of animals exposed to the heavy metals, a decrease in expression of Hsp70 was sometimes detected. Thus Hsp analysis by Western blot in L. terrestris tissues and particularly in the midgut/intestine proved to be a suitable and sensitive assay for adverse effects in earthworms and showed a good level of reproducibility despite some individual variations. The use of pristine/nonexposed animals transposed into contaminated environments as in the present study should therefore be of high ecological relevance. Induction of Hsp70 in earthworms should represent not only a good wide-spectrum biomarker of exposure but also a biomarker of effect since known toxicants altered gene expression in tissues of these animals, as contrasted with a simple accumulation of Hsp. Hence, the detection of Hsp70 in earthworms can constitute an early-warning marker for the presence of potentially deleterious agents in soils, with L. terrestris in particular and earthworms in general acting as potential sentinel animal species.
... protozoaire, nématodes…) peuvent aussi décliner avec la digestion mais ils ne constituent probablement qu'une part mineure de l'assimilation des lombriciens contrairement aux microorganismes (Larsen et al., 2016;Schmidt et al., 2016). De nombreuses enzymes ont été détectées dans le tube digestif des lombriciens (cellulases, chitinases, phosphatases, …) participant à la libération de molécules à faible poids moléculaire et donc de nutriments dont une partie peut être assimilée par les lombriciens (Tracey, 1951;Laverack, 1963;Prentø, 1987). L'origine de ces enzymes fait toujours débat entre une production directe par les lombriciens ou une synthèse par les microorganismes ingérés Egert et al., 2004;Drake and Horn, INTRODUCTION 26 Les excrétions des anéciques se font sous différentes formes : le mucus, l'urine et les déjections. ...
... Due to the high concentration of microorganisms present in the digestive tract of earthworms, there is a high concentration of enzymes. Thus, gut content of L. terrestris contains several enzymes involved in several biogeochemical cycles such as C, N and P (Laverack, 1963;Prentø, 1987;Urbasek, 1990). With all of these digestive enzymes, their presence seems to decrease along the gut transit with some exception (among others, phosphorylase and glutamate dehydrogenase) (Jeuniaux, 1969;Tillinghast and Macdonnell, 1973). ...
La décomposition des litières est un processus clé du fonctionnement du sol contribuant à de nombreux services écosystémiques. En climat tempéré, les lombriciens en interaction avec les micro-organismes du sol, contribuent significativement à ce processus. Cependant, les connaissances sur les lombriciens ciblent le plus souvent les trois catégories écologiques selon lesquelles ils sont définis : les épigés, les endogés et les anéciques. Les anéciques sont très répandus dans les sols tempérés, constituent la majeur partie de la biomasse lombricienne et interviennent dans la décomposition des litières. Plusieurs études ont observé des traits comportementaux, morphologiques et physiologiques distinguant deux sous-catégories au sein des anéciques : les épi-anéciques et les anéciques stricts. Le premier objectif de ce travail de thèse était de vérifier si cette distinction avait une réalité dans le cadre du processus de décomposition des litières. En conditions contrôlées, nous avons évalué (i) le rôle des principales espèces anéciques dans le processus de décomposition, (ii) l’impact de ces espèces sur les communautés de microorganismes et (iii) les activités enzymatiques des microorganismes du sol et (vi) l’impact des interactions entre espèces anéciques sur le processus de décomposition. A partir d’observations sur le terrain, le second objectif de ce travail de thèse était de définir les règles d’assemblages des communautés lombriciennes en prairie compte-tenu de leurs rôles majeurs dans divers processus du sol. Les résultats obtenus ont confirmé la distinction entre lombriciens anéciques stricts et épi-anéciques : les épi-anéciques étant les seuls à contribuer au processus de décomposition des litières et celui-ci étant corrélé à la biomasse individuelle moyenne de chaque espèce. Cette contribution passe par une plus forte stimulation des activités enzymatiques du sol, indépendamment des espèces considérées. En revanche, contrairement aux bactéries, les communautés fongiques du sol dépendent de l’espèce épi-anécique avec laquelle ils interagissent. Ce travail met également en évidence que l’abondance, la biomasse et la diversité des communautés lombriciennes des sols prairiaux sont régulées par différents filtres environnementaux dont la diversité du paysage. Cette thèse met en évidence que les deux sous-catégories écologiques au sein des anéciques ont des rôles différents sur le processus de décomposition des litières et qu’elles contribuent donc à des services écosystémiques fournis par le sol de manière différenciée.
... The general occurrence of the chloragogenous tissue in oligochaetes, their close connection with the body fluids and with the gut, and their frequently voluminous extent strongly suggest that this tissue plays an essential role in the life of oligochaete worms (Fischer and Molnár, 1992). Elsewhere, the functions of chloragogenous tissue have been documented to include probable storage cells for glycogen, lipids, and phosphate, and play a critical role in the removal of superoxide and hydrogen peroxide arising from the interaction of molecular oxygen with the blood protein (Prentø, 1987). As such the depletion of this tissue may have implications for outcomes of oxidative stress in this species. ...
This study evaluated Acetic acid (AA) and Benzoic acid's (BA) acute and sublethal toxicity by observing mortality, behavioral responses, and changes in the levels of oxidative stress enzymes in Tubifex tubifex. Exposure-induced changes in antioxidant activity (Catalase, Superoxide dismutase), oxidative stress (Malondialdehyde concentrations), and histopathological alterations in the tubificid worms were also noted across exposure intervals. The 96 h LC50 values of AA and BA to T. tubifex were 74.99 and 37.15 mg/l, respectively. Severity in behavioral alterations (including increased mucus production, wrinkling, and reduction in clumping) and autotomy showed concentration-dependent trends for both toxicants. Although histopathological effects also showed marked degeneration in the alimentary and integumentary systems in highest exposure groups (worms exposed to 14.99 mg/l for AA and 7.42 mg/l for BA) for both toxicants. Antioxidant enzymes (catalase and superoxide dismutase) also showed a marked increase of up to 8-fold and 10-fold for the highest exposure group of AA and BA respectively. While species sensitivity distribution analysis revealed T. tubifex as most sensitive to AA and BA compared to other freshwater vertebrates and invertebrates, General Unified Threshold model of Survival (GUTS) predicted individual tolerance effects (GUTS-IT), with slower potential for toxicodynamic recovery, as a more likely pathway for population mortality. Study findings demonstrate BA with greater potential for ecological effects compared to AA within 24 h of exposure. Furthermore, ecological risks to critical detritus feeders like T. tubifex may have severe implications for ecosystem services and nutrient availability within freshwater habitats.
... Interestingly, gastrointestinal secretion of an important group of pesticide-detoxifying enzymes called carboxylesterases (EC 3.1.1.1) was demonstrated by Sanchez-Hernandez et al. (2009), corroborating past findings in L. terrestris (Prentø 1987) and other invertebrate species (Mommsen 1978, Geering and Freyvogel 1975, Turunen and Chippendale 1977, Turunen 1978. Carboxylesterases activity play an important role in the metabolism of xenobiotics in mammals (Hatfield et al. 2016). ...
... Similarly, an inverse relationship was observed between body size and plasma esterase activities in birds (Roy et al., 2005) and amphibians (Lajmanovich et al., 2008). Dietary lipids are also important inducers of CbE activity in mammals (van Lith et al., 1992), passerine birds (Ríos et al., 2014 ), and some invertebrate species (Mommsen, 1978; Prento, 1987); accordingly, this esterase activity seems to be actively involved in lipid metabolism. To our knowledge, there are no data on the response of CbE and AChE activities to OP exposure in animals fed different diets. ...
An outdoor microcosm was performed with tadpoles (Rhinella arenarum) exposed to 125 μg L À 1 chlor-pyrifos and fed two types of food, i.e., lettuce (Lactuca sativa) and a formulated commercial pellet. Acetylcholinesterase (AChE) and carboxylesterase (CbE) activities were measured in liver and intestine after 10 days of pesticide exposure. Non-exposed tadpoles fed lettuce had an intestinal AChE activity almost twofold higher than that of pellet-fed tadpoles. No significant differences were observed, however, in liver AChE activity between diets. Likewise, intestinal CbE activity – measured using two substrates, i.e. 1-naphthyl acetate (1-NA) and 4-nitrophenyl valerate (4-NPV) – was higher in tadpoles fed lettuce than in those fed pellets. However, the diet-dependent response of liver CbE activity was opposite to that in the intestine. Chlorpyrifos caused a significant inhibition of both esterase activities, which was tissue-and diet-specific. The highest inhibition degree was found in the intestinal AChE and CbE activities of lettuce-fed tadpoles (42–78% of controls) compared with pellet-fed tadpoles (o 60%). Although chlorpyrifos significantly inhibited liver CbE activity of the group fed lettuce, this effect was not observed in the group fed pellets. In general, intestinal CbE activity was more sensitive to chlorpyrifos inhibition than AChE activity. This finding, together with the high levels of basal CbE activity found in the intestine, may be understood as a detoxification system able to reduce intestinal OP uptake. Moreover, the results of this study suggest that diet is a determinant factor in toxicity testing with tadpoles to assess OP toxicity, because it modulates levels of this potential detoxifying enzyme activity.
... However, changes occurred in the chloragogenous tissue (vacuolization and hypertrophy). The oligochaete chloragenous tissue is described in the literature as functionally analogous to the liver in vertebrates [29,30]. This was in agreement with observations in a study on Danio rerio exposed to phenanthrene, showing vacuolization and hypertrophy of hepatocytes [31]. ...
Polycyclic aromatic hydrocarbons (PAHs) continue to cause environmental challenges due to their release in the environment by a great variety of anthropogenic activities and their accumulation in soils. Here we studied the toxicological effect of the model PAH phenanthrene on the soil invertebrate model Enchytraeus crypticus at the individual, tissue, and molecular level. Animals were exposed for 2 and 21 d to phenanthrene concentrations corresponding with the (previously estimated) 3-week EC10 and EC50 for effects on reproduction. Gene expression profiling did not reveal a typical phenanthrene-induced biotransformation signature, as it usually does in arthropods and vertebrates. Instead, we observed only general metabolic processes to be affected after 2 d of exposure, such as translation and ATP synthesis-coupled electron transport. Histological sections of tissues of 2-day exposed animals did not show any deviations from the control situation. In contrast, prolonged exposure up to 21 d showed histopathological effects: chloragogenous cells were highly vacuolated and hypertrophic. This was corroborated by differential expression of genes related to immune response and oxidative stress at the transcriptomic level. The data exemplify the complexity and species-specific features of PAH toxicity among soil invertebrate communities, which restricts read-across and extrapolation in the context of soil ecological risk assessment. This article is protected by copyright. All rights reserved
... Chloragogenous tissue surrounding the intestine, where most of the key metabolic processes occur (Prentø, 1987), also presented significant changes in treated animals. In particular, a significant lysosomal accumulation of lipofuscin (end-products of membrane lipid peroxidation e Terman and Brunk, 2004;Viarengo and Nott, 1993) and of neutral lipids (due to unbalanced fatty acid metabolism e Lüllman-Rauch, 1979) was induced in the chloragocytes of worms exposed for 28 d only to some concentrations. ...
... In the post-clitellar region of the alimentary canal, i.e. the intestine, takes place most of the digestion and absorption of food materials. The intestine is surrounded by the chloragogenous tissue that plays several functions, including storage of nutrients and excretion of the waste products of cell catabolism (Prentø, 1987). It has been demonstrated that diverse xenobiotics are also stored in this tissue (Fischer and Horvath, 1976;Morgan and Morgan, 1989;Cancio et al., 1995;Stürzenbaum et al., 2004). ...
In this study, a battery of biomarkers was utilised to evaluate the stress syndrome induced in the earthworm Eisenia andrei by exposure to environmentally realistic concentrations of benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) in OECD soil. The set of tests was then employed to assess the toxicity of field soils contaminated with organic xenobiotic compounds (such as PAHs, dioxins and PCBs). The results highlighted an impairment of immune and metabolic functions and genotoxic damage in worms exposed also to lower bioavailable concentrations of toxic chemicals. Multivariate analysis of biomarker data showed that all different contaminated soils had a detrimental effect on the earthworms. A separation between temporal and concentration factors was also evident for B[a]P and TCDD treatments; and field contaminated soils were further differentiated reflecting a diverse contamination. Multivariate analysis also demonstrated that lysosomal membrane stability can be considered a prognostic indicator for worm health status.
... In the post-clitellar region of the alimentary canal, i.e. the intestine, takes place most of the digestion and absorption of food materials. The intestine is surrounded by the chloragogenous tissue that plays several functions, including storage of nutrients and excretion of the waste products of cell catabolism (Prentø, 1987). It has been demonstrated that diverse xenobiotics are also stored in this tissue (Fischer and Horvath, 1976;Morgan and Morgan, 1989;Cancio et al., 1995;Stürzenbaum et al., 2004). ...
An immunohistochemical method using antibodies against polycyclic aromatic hydrocarbons (PAHs) and dioxins was developed on frozen tissue sections of the earthworm Eisenia andrei exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50ppm) and 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) (0.01, 0.1, 2ppb) in spiked standard soils. The concentrations of B[a]P and TCDD in E. andrei exposed to the same conditions were also measured using analytical chemical procedures. The results demonstrated that tissues of worms exposed to even minimal amount of B[a]P and TCDD reacted positively and specifically to anti-PAHs and -dioxins antibody. Immunofluorescence revealed a much more intense staining for the gut compared to the body wall; moreover, positively immunoreactive amoeboid coelomocytes were also observed, i.e. cells in which we have previously demonstrated the occurrence of genotoxic damage. The double immunolabelling with antibodies against B[a]P/TCDD and the lysosomal enzyme cathepsin D demonstrated the lysosomal accumulation of the organic xenobiotic compounds, in particular in the cells of the chloragogenous tissue as well as in coelomocytes, involved into detoxification and protection of animals against toxic chemicals. The method described is timesaving, not expensive and easily applicable.
... The general occurrence of the chloragogenous tissue in oligochaetes, their close connection with the body fluids and with the gut, and their frequently voluminous extent suggest that this tissue plays an essential role in the life of oligochaete worms. Based on data from other authors and on his own results Prento ( 1987) summarized the functions of chloragogenous tissue. He concluded that chloragocytes (I) are probably mainly storage cells for glycogen, lipids and phosphate; (2) are important in the removal of superoxide and hydrogen peroxide arising from the interaction of molecular oxygen with the blood protein, which, most likely, is also synthesized by chloragocytes; (3) are only to a small extent involved in intermediary metabolism, except possibly in lipid metabolism; (4) and may accumulate various foreign substances. ...
Environmental factors affecting the chloragogenous tissue of the manure worm, Eisenia fetida and other oligochaetes were surveyed. Chloragocytes might be affected-detectable by karyometric methods—by hydration, desiccation, cold stress, hypoxia, saline load, or by diverse xenobiotics. Chloragocyte depletion may be stimulated by some pesticides. Paraquat toxication can cause an almost full depletion of the chloragogenous tissue as we observed, followed in survivors by a renewal of this tissue. Experimental cold stress induced a marked increase of the Carnoy-insoluble pigmentation of the chloragosomes in juvenile worms. Moderate accumulation of such pigments could be observed on the effect of saline load. The elemental composition of chloragosomes might be markedly influenced by some environmental factors, including heavy metals. Experimentally-induced alterations of the chloragogenous tissue may contribute to the adaptation of the earthworms to variable conditions of their environments.
... The role of the chloragogenous tissue in hemoglobin synthesis has been suggested (Lindner, 1965;Fischer and Horváth, 1978;Prentø, 1987;Lee et al., 2005), and it probably synthesizes metallothioneinlike proteins (Ireland, 1983;Morgan et al., 1989). Mangum and Dales (1965) showed that there is a quantitative difference between annelid and vertebrate hemoprotein biosynthesis in that large amount of porphyrin by-products formed in the hemopoietic tissue of annelids and these by-products were accumulated in granular structures of the cytoplasm. ...
The cytochemical and functional characteristics of chloragocytes of both 'control' and cold-stressed Eisenia fetida were examined. Flow cytometry revealed the heterogeneity of chloragocytes: the first group was characterized by low, the second one by high acid phosphatase (AcP) content. In 'control' animals the former, in cold-stressed ones the latter type were the dominant form. The elevated AcP-activity correlated with the accumulation of autophagic vacuoles (AVs) in chloragocytes. Both AVs and all small chloragosomes showed high AcP activity, while most of the large chloragosomes did not display any. Most 'control' granules (0.75-1.25 μm) contained high amounts of Ca and P, with less and variable quantities of S, Cl, K, Fe and Zn. Small chloragosomes with low Ca and P concentrations were seldom found. In cold-stressed animals the number of small granules (0.25-0.75 μm) increased up to 40% of total population. Their Ca and P contents were significantly lower; S and Fe concentrations were higher than those of large chloragosomes (1.0-1.5 μm). Our results prove that the formation and elemental composition of chloragosomes can be influenced by environmental stressors and suggest that the mature chloragosomes are tertiary lysosomes and their formation is coupled to autophagocytosis.
... Multiples CE isozymes are present in earthworms (Haites et al., 1972), and they show a tissue-dependent sensitivity to OP insecticides such as chlorpyrifos-oxon (CPOx) (Sanchez-Hernandez and Wheelock, 2009). These esterases are particularly abundant in the earthworm gut (Prento, 1987) Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c b p c documented in the gastrointestinal tract of L. terrestris (SanchezHernandez et al., 2009). Despite these investigations, more laboratory and field studies are still necessary to understand the role of CEs in pesticide toxicity in earthworms, as well as to investigate their usefulness as biomarkers of pesticide exposure in biomonitoring programs. ...
Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil.
... Many reports have been published on the localization of digestive enzymes in various invertebrate taxa such as trematoda (Bogitsh and Dresden 1983), nemertini (Gibson and Egan 1976), sipunculida (Michel and DeVillez 1984 ), gastropoda (Onishi et al. 1985), bivalvia (Palmer 1979 ), oligochaeta (Prento 1987), polychaeta (Michel and DeVillez 1979), insecta (Lehane 1976 ), arachnida (Ludwig and A1- berti 1988) or echinodermata (Tokin and Filimonova 1977). As in crustaceans, the site of biosynthesis of digestive enzymes often was deduced from enzyme activities in tissue homogenates or evaluated by histochemistry using synthetic substrates. ...
For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.
... These findings suggest that ALP functions, at least in part, similarly in mammals and E. japonensis. ALP activity in the digestive tract has been observed in other invertebrates, including oligochaete annelids (Gelder, 1984;Prentø, 1987), insects (Eguchi, 1995;Funk, 2001), and ascidians (Kawamura and Fujiwara, 1993), and ALP activity in excretory organs has been observed in the Malpighian tubule, the insect counterpart of the kidney (Yang et al., 2000;Yi and Adams, 2001). In E. japonensis, ALP activity was also observed in various other tissues. ...
The fragmenting potworm Enchytraeus japonensis (Oligochaeta, Annelida) reproduces asexually by dividing the body into several fragments that then regenerate to complete individuals in 4-5 days. Such large-scale regeneration, however, occurs only in some invertebrates. To better our understanding of why regeneration is so limited in many animals, despite their ability to undergo embryonic development from the single cell of a fertilized egg, comparisons were made between regeneration and embryonic development of E. japonensis by using two methods: histochemistry for alkaline phosphatase (ALP) and immunohistochemistry with an antibody against acetylated tubulin that visualizes nervous system development. The analyses revealed that both ALP expression patterns and central nervous system development differ between embryogenesis and the regeneration, suggesting that regeneration is not a simple reiteration of embryogenesis but involves different regulatory mechanisms. The study provides a basis for the elucidation of mechanisms that are unique and crucial to regeneration.
... Studies of earthworm physiology have included characterization of gut enzymes (16,24), yet the source and degradative capability of the enzymes are debatable. The possibility that some enzymes, especially cellulases, might be of microbial origin has been considered previously (10,15,31). ...
Viable bacteria were found to coexist with developing embryos in egg capsules (cocoons) of the earthworm Eisenia fetida. Earthworms were reared under standardized conditions, and bacterial densities were measured in distinct batches of cocoons collected weekly for 10 weeks. Cocoons weighing 12 mg contained a mean viable bacterial population of approximately 10 CFU/g of cocoons. No difference was found in viable counts obtained from cocoons incubated at 15 degrees C and cocoons incubated at 24 degrees C. Viable bacterial numbers increased with cocoon age, while acridine orange direct counts of microbial cells were stable at approximately 10 cells per g of cocoons. Bacteria isolated from cocoons were used to develop antisera in rabbits for the production of strain-specific fluorescent antibodies. Fluorescent antibody and selective plating techniques were used to monitor populations of these bacteria in earthworm bedding and to determine whether cocoons acquire bacteria from the environment in which they are formed. Cocoon isolates were readily recovered from cocoons formed in inoculated bedding at densities of 10 CFU/g of cocoons. Bradyrhizobium japonicum USDA 110 and UMR 161 added to bedding were also recovered from cocoons, but at lower densities than cocoon isolates. Escherichia coli K-12(pJP4) inoculum was recovered from bedding but not from cocoons. The bacterial complement of Eisenia fetida cocoons is affected by inoculation of selected bacterial isolates in the worm growth environment.
A major uptake route of nanoparticles (NPs) occurs via the gastrointestinal (GI) tract. When GI tract cells are exposed, NPs cytotoxic effects are observed that subsequently adversely affect the GI tract morphology and have consequences for the whole organism. The aim of this study was to understand the mechanism of effects caused by ZnO-NPs compared to Zn ions on the earthworm Eisenia andrei.
The following aspects of individually exposed earthworms were investigated: 1) qualitative structural alterations in the gut epithelium and chloragogen cells of the GI tract, 2) quantitative changes within chloragogen tissues after 48 h of exposure (using morphometric analysis), and 3) the ADP/ATP ratio in homogenized tissue of the whole organism after 21 days of exposure to contaminated soil (contamination phase) followed by 14 days of elimination in clean soil (decontamination phase) to identify possible recovery.
Both ZnO-NPs and Zn ions adversely affect the gut epithelium and chloragogen tissue of earthworms after 48 h of exposure to contaminated soil. Morphometric measurements revealed that the proportions of debris vesicles in the chloragocytes were significantly lower in worms exposed to ZnO-NPs than in worms exposed to Zn ions. Moreover, numerous spherite granules were observed in the chloragocytes of ionic Zn-treated worms, but not the ZnO-NPs-treated worms, suggesting differential regulation of these Zn forms. The Zn cytotoxic effect was not reflected in ADP/ATP ratio measurements. Our study provides new insights into nano-specific effects that are distinctive from ion regulation inside the GI tract and furthers our understanding of the relationship between effects at the cellular and whole-body levels.
Knowledge on the nutritional ecology of enchytraeids is scarce. Therefore, laboratorial investigations were undertaken to study uptake, fate and digestive processes of different food sources (Penicillium sp., Bacillus cereus var. mycoides, Sambucus nigra, rolled oats, soil) by Enchytraeus coronatus. Feeding behaviour, cocoon production and mortality of the worms were observed with the aid of a light microscope. Ultrahistological investigation of the gut and its luminal contents were used to recognize; (1) the fate of the food passing the gut, (2) alteration of the gut tissues due to the diet. The number of cocoons and adult worms depended on the diet and decreases from rolled oats < Sambuca nigra < Bacillus cereus < Penicillium. Remnants of food were generally rare but nearly all offered diets except Penicillium were found in the gut. Because of the small amount of remnants in the gut, preoral digestion must be assumed. Alteration of the gut epithelia resulting from food quality could not be observed. The only prominent alteration was found in the chloragogenous tissue. Feeding with rolled oats results in a high number of fat vesicles within the chloragogenous cells. The results show that enchytraeids have a widespread nutrient spectrum and are possibly not a member of the group of primary decomposer.
The induction of the antioxidant enzymes, catalase and superoxide dismutase (SOD) was investigated for potential use as biomarkers for oxidative stress generated by heavy metals and paraquat in earthworms. The species Eisenia veneta and E. fetida were exposed to the heavy metals Zn, Cu, and Hg and the herbicide paraquat for 6 days in an artificial liquid medium. Eisenia veneta was also exposed to the same heavy metals in artificial soil for 14 days. We found that the catalase and SOD activity were not induced in the worms by any of the oxidants tested, neither for 6 days exposure in liquid medium or 14 days exposure in soil. The catalase activity showed large inter-species difference, as the activity was 6-8 times higher in E. veneta than in E. fetida. The SOD activity did not differ significantly between the two species, but most of the SOD activity in these worms was due to the non-inducible Cu, Zn-SOD class, with only 4-12 % Mn-SOD activity present. The results indicate that catalase and SOD are not suitable as biomarkers of oxidative stress generated by heavy metals or paraquat in earthworms.
Since their heterotrophic origin, animals have been faced with a continual input of foreign compounds, so-called xenobiotics, from their food sources, e.g. polybromomethanes and alkyl halides in seaweeds (Gschwend et al. 1985) and defensive toxins in other animals (Bakus and Kawaguchi 1984); and from the attacks of predators, e.g. venoms of certain gastropods and cephalopods (Fange 1984). For example, “animal-plant warfare” is considered to be a major selective pressure in driving the evolution of some of the gene families of the biotransformation enzyme, cytochrome P-450 (Nebert et al. 1989b). Over time, additional sources of xenobiotics have included erosion of hydrocarbon-containing shales, and oil seepage from natural reservoirs (started at least 100,000 years ago) (Farrington 1985), hostile environments such as the high sulphide levels around deep sea hydrothermal vents (Vetter et al. 1987), and, in more recent years, man’s multifarious industrial and other activities (Mix 1984). Central to the defense against such an enormous and diverse number of potentially toxic compounds is an impressive array of enzymes, which function ideally to detoxify and eliminate xenobiotics from an organism. The biological significance of biotransformation enzymes is increased by the inducibility of some of them by xenobiotics, and by their metabolism of certain xenobiotics to molecular species more toxic, mutagenic or carcinogenic than the parent compound. Elaboration of the qualitative and quantitative aspects of organic xenobiotic metabolism in marine invertebrates, and the function and regulation of the enzymes involved, is important for several reasons, viz. predicting and modelling the fate and toxicity of xenobiotics in marine organisms and ecosystems (e.g. Harris et al. 1984); development of specific indices of biological effect for use in pollution monitoring and impact assessment (e.g. Malins et al. 1985; Kleinow et al. 1987; Payne et al. 1987; Bayne et al. 1988); and understanding the evolutionary relationships of the biotransformation pathways in different phylogenetic groups, and the use of the pathways in invading, or exploiting, ecological time and space to create new niches.
The perivascular tissue of Sabella pavonina, a layer of brown cells covering ventral and lateral vessels, is characterized by three types of distinct inclusions in the cells: chlorocruorin vacuoles, ferritin particles and granules, and vacuoles containing both chlorocruorin and ferritin. The crystallization of chlorocruorin in trans-Golgian vesicles and the gathering of these into vacuoles suggests that this tissue is involved in the chlorocruorin synthesis and indicates that the terminal stage of this synthesis occurs in the Golgi apparatus. The basolateral exocytosis of this respiratory pigment in intercellular spaces and its transit to the lumen of the vessels are revealed. Alternating changes in chlorocruorin and ferritin contents of chloragocytes linked to ultrastructural variations in these cells, as observed in many sabella, led to the conclusion that the perivascular tissue play a role in both chlorocruorin synthesis and temporary iron storage.
Our aim was to determine haemoglobin content in earthworm populations from sites with different amounts of soil pollution. The study was done on Lumbricus terrestris specimens from four sites: the median strip of a busy street in Cracow; a city park; forest about 30 km from Cracow (100 m from a low-traffic local road); and a forest on calcareous soil about 30 km from Cracow (distant from traffic). There were statistically significant differences in haemoglobin content between study sites. The lowest amount of haemoglobin was found in earthworms from the median strip (2.19 g 100 ml−1), the highest in those from the ‘cleanest’ forest on calcareous soil (3.18 g 100 ml−1). The highest lead concentration was found in specimens from the median strip (12.3 μg g−1) and only trace amounts were found in those from the forest on calcareous soil. There was a negative correlation between haemoglobin and lead concentrations in earthworms. Species composition, density and biomass of earthworm populations at the four sampling sites as well as the body mass of adult individuals of L. terrestris were estimated. No correlation was found between any of these variables and the extent of exposure to traffic pollution.
1.1. Intestines of fresh and dehydrated-starved L. terrestris were compared to tissue and anterior-posterior distribution of glutamate dehydrogenase (GDH) and other mitochondrial or cytosol dehydrogenases.2.2. For any dehydrogenase, including GDH, practically all the activity was in the gut epithelium. This distribution of GDH supports Tillinghast (1967, 1968) as to the excretory route for ammonia.3.3. While the distributions of the marker dehydrogenases were reasonably uniform along the intestine, the GDH activity was predominantly (80–90% of the total activity) in the last third of the mid-intestine, indicating a true physiological differentiation of the midgut tube. The GDH activity of the typhlosole was about two times the activity in the peripheral epithelium. The GDH distribution was independent of the physiological state of the worm.4.4. From the distribution of GDH it follows that the mid-intestine, immediately before the hindgut, is the main region both for amino acid uptake and catabolism. As regards amino acids, it typifies the primitive digestive tube by having both the absorptive and the liver functions.
Solar radiation leads to hydrogen peroxide (H202) accumulation in shallow intertidal sur-face waters dunng daytime tidal emersion periods. The lugworm Arenjcola marina irrigates its sedi-mentary tubes with surface water containing variable H 2 0 2 concentrations. We studied aspects of the antioxidant status of the intertidal polychaete A. marina in response to seasonal variations of oxidative stress in its environment. Antioxidant enzyme activities [superoxide dismutase (SOD), glutathione reductase (GR) and catalase] and v~tamin E were concentrated chiefly In the chloragog and to a lesser extent in body wall tissue. Response to experimental H 2 0 2 exposure (5 1.lmol I-') was confined to the chloragog tissue and consisted of elevated catalase activity. On a subcellular level, the major part of enzymatic antioxidants examined was found in the cytosolic (77 % of SOD activity, 60% of GR activity, 41% of catalase activity, 87% of total glutathione) and the peroxisomal (56% of catalase activity) fractions, whereas the same antioxidants were virtually absent in mitochondria. Gradual acclimation of winter A. marina (5°C) to higher ambient temperatures (20°C) over 20 d resulted in a doubling of chloragog SOD activities, while catalase activity was not affected. Elevated in situ concentrations of photoproduced H202 during summer (1.7 pm01 1-l) coincided with a significant increase of chloragog catalase activities in young (winter-317.97 + 78.3 U mg-' protein, summer: 783.41 + 192.7 U mg-' pro-tein, means * SD) and in adult (winter: 480.09 * 160.1 U mg-' protein, summer: 1165.5 * 207.5 U mg-' prote~n) lugworms. Seasonal differences in the SOD activities of adult worms (winter: 16.06 i 4.69 SOD units mg-' protein; summer: 23.29 * 3.31 SOD units mg-' protein) were attributed to the elevated sedi-ment temperatures in intertidal areas during summer.
Purpose
The use of only one or a few species—representing an entire taxon—in ecotoxicological standard tests poses risk of underestimating the impact of toxicants on the environment. In earthworm ecotoxicity tests, the species Eisenia fetida or Eisenia andrei are commonly used, and there is evidence that these species respond relatively insensitive towards environmental pollution. With the present study, we wanted to evaluate the risk of underestimating effects of the insecticide imidacloprid in soil organisms by comparing E. fetida with two other earthworm species (Aporrectodea caliginosa and Lumbricus terrestris) regarding their sensitivities towards soil contaminated with this widely used insecticide.
Materials and methods
In laboratory experiments, the specimens were individually exposed to various concentrations of the pesticide (0.2, 0.66, 2 and 4 mg kg−1 dry weight (DW)) for 1, 7 and 14 days. Afterwards, histopathological changes in the midgut, chloragogenous tissue and skin, as well as body mass changes, were assessed.
Results and discussion
While significant changes in body mass in E. fetida and A. caliginosa occurred after exposure to imidacloprid concentrations as low as 0.2 (7 days) and 0.66 mg kg−1 DW (14 days), significant body mass changes in L. terrestris observed to 2 and 4 mg kg−1 DW, for 7 and 14 days of exposure, respectively. The histopathological examinations revealed that significant cellular changes already occurred after 24 h exposure to the lowest test concentrations in all species, but the degree of detrimental effects as well as species-specific differences were dependent on the monitor tissue. In general, E. fetida seemed to be more sensitive than L. terrestris concerning cellular alterations, but the hierarchy in species-specific differences was less obvious than for body mass change.
Conclusions
Even if E. fetida proved to be the most sensitive species in this study, general differences in sensitivity make evident that always a range of species—being representatives of an animal taxon—in ecotoxicological tests should be tested in order to avoid underestimations of effects. In the case of testing only one species, an increase of safety factors should be considered. Since effects already occurred at environmentally relevant concentrations, the use of imidacloprid in agriculture might be of great concern.
1.1. Chloragogenous, extravasal, intravasal and botryoidal tissues occur alternatively in various types of annelids. These tissues are sources of extracellular respiratory pigments. Considering their peritoneal origin, their structural and functional similarities we propose the term “chloragogenous-like tissues” for their common designation.2.2. The synthesis of haem-compounds plays a significant role in the development of the structural and functional characteristics of the chloragogenous tissues. Haemoglobin is released into the blood plasma but the haem-enzymes catalase and peroxidase accumulate into the chloragocytes, moreover haem by products and damaged haem-compounds may be involved in the formation of chloragosomes.3.3. Chloragocytes are in some way involved in the immune defence reactions.4.4. We conclude that the chloragogenous-like cells are specific myelo-erythroid cells of the annelids.
Phosphotriesterase (PTE) receives attention because it seems to be associated with the detoxification of organophosphorous pesticides and organophosphate resistance mechanism. In order to understand the biodegradation of phosphotriester pesticides and its significance in the earthworm, a major non-target animal of pesticides, selected properties of phosphotriesterase activity derived from the crude extract of Eisenia andrei were investigated. PTE activity appeared to be primarily localized in intestinal tissues. The highest level of PTE activity was found in epithelial tissue. The native molecular weight of earthworm PTE was 260 kDa and the isoelectric point was approximately 4. The optimal pH was approximately 9. The earthworm PTE had a substrate affinity for paraoxon with Km value in the millimolar range. The presence of EGTA and EDTA completely abolished the activity and replacement of Ca2+ ion restored activity to greater than 95%, suggesting that Ca2+ ion is essential to maintain the activity.
1.1. Guts of fresh and dehydrated-starved L. terrestris were compared as to tissue and anterior-posterior distribution of arginase, OCT (ornithine carbamoyl phosphate transferase) and AS (arginosuccinase).2.2. In fresh worms the anterior two thirds of the midgut was practically devoid of arginase activity, while in starved worms this region exhibited a high arginase activity. Both fresh and starved worms had high arginase activity in the posterior part of the midgut and in the hindgut, and this arginase activity did not increase during starvation. The distribution of AS and OCT was relatively uniform along the gut regardless of state of the worm.3.3. The main site of the constant posterior arginase activity was the intestinal epithelium, so part of the posterior midgut epithelium may possibly carry out both ammonia (Prentø P. (1989) Comp. Biochem. Physiol. B., in press) and urea production. OCT and AS were always present in both chloragog tissue and epithelium.4.4. Starved worms had about four times higher arginase, AS and OCT activities than in fresh worms. Most of the arginase activity (about 70%) was in the chloragog tissue, while the remaining activity was in the intestinal epithelium. This distribution supports Tillinghast (Tillinghast E. K. (1987) J. exp. Zool166, 295–300; and (1986) Comp. Biochem. Physiol.24B 621–623.) as to the excretory route for urea. About 66% of the gut OCT and AS activities were found in the chloragog tissue.5.5. The distribution of arginase suggests that both for the intestinal epithelium and for the chloragog tissue high external ammonia levels induce arginase synthesis. The distribution of OCT and AS suggest that arginine synthesis is a property of both the chloragog tissue and the intestinal epithelium.
Expression of alkaline phosphatases in developing embryo and mature stages of the earthworm, Eisenia andrei was investigated. The embryonic stages examined in this study appeared to have only one slow-moving form of alkaline phosphatase which had a different mobility from the intestinal alkaline phosphatases of the mature worm, suggesting that intestinal alkaline phosphatases of embryos may be different from mature forms. A surge in alkaline phosphatase activity after hatching is consistent with the expression of mature forms of intestinal alkaline phosphatase and this increase would be associated with postnatal differentiation of the intestine.
1. Activities and genetic banding patterns of 36 isozymes in carbohydrate metabolism were detected by spectrophotometry and starch-gel electrophoresis, respectively, in the earthworm Eisenia fetida. 2. Polymorphisms were not distributed randomly among metabolic pathways, activity levels, or gene copy numbers. 3. In glycolysis and the Krebs cycle, 16% of the loci were polymorphic and polymorphisms occurred only when multiple copies of the loci existed. 4. In other pathways, 45% of the loci were polymorphic and the distribution of polymorphisms was independent of gene copy number. 5. Polymorphisms may affect metabolic phenotypes and natural selection may have led to conserved biochemical activity in glycolysis and the Krebs cycle.
1. Earthworms can hydrolyze di-(2-ethylhexyl) phthalate (DEHP) to mono-2-ethylhexyl phthalate (MEHP) and phthalic acid (PA). 2. They apparently cannot produce the side-chain-oxidized derivatives of MEHP that constitute the major DEHP metabolites in higher animals. 3. With the assistance of intestinal bacterial Pseudomonas, the worm-derived PA is degraded through protocatechuic and beta-carboxymuconic acids to CO2. 4. There is an indication of a second pathway for degradation of PA leading through benzoic acid.
The cytochemical localization of the lysosomal marker enzyme acid phosphatase was studied in the chloragogenous tissue of earthworms. The Gomori lead technique and the cerium capture technique were utilized. Both techniques demonstrated the chloragosomal location of this enzyme. Only a small proportion of chloragosomes presented reactivity, which suggests that these organelles are distinctly heterogeneous. The reaction product was localized in the periphery of chloragosomes, suggesting a membrane-bound compartmentalization of acid phosphatase. In addition, degenerating mitochondria and membrane whorls were observed in some chloragosomes, indicating the possibility that these organelles perform autophagosomal functions.
Chloragocytes were isolated from the earthworm species Lumbricus terrestris. After mechanical dissociation and sedimentation through Percoll, a highly purified fraction of viable chloragocytes was obtained. The isolated chloragocytes accumulated the vital dye neutral red and reduced the tetrazolium dye MTT, thereby indicating cellular integrity. Time of flight flow cytometric analyses revealed a main population of large and highly granulated cells in the 30-33 microm size range. Hydrolase measurements showed that beta-D-N-acetyl-glucosaminidase and acid phosphatase exhibited the highest activities (146.6 and 24.9 mU/mg of protein, respectively), possibly indicating a major role for these 2 hydrolases in the physiological function of chloragocytes. In contrast, other acid hydrolases such as beta-D-galactosidase and beta-D-glucuronidase had specific activities of respectively 26 and 182 times lower than the glucosaminidase. The specific activity of the membrane-bound alkaline phosphatase was comparable to that of its acid counterpart (18.9 vs. 24.9 mU/mg of protein, respectively) and this level of activity may show an important trans-membrane activity in chloragocytes. The cytoplasmic and mitochondrial enzyme isocitrate dehydrogenase had a level of activity comparable to that of the exclusively cytoplasmic enzyme lactate dehydrogenase (6.6 vs. 8.1 mIU/mg of protein, respectively). When L. terrestris chloragocyte homogenates were separated on Percoll, results showed that hydrolases and dehydrogenases were mainly associated with the lighter materials that remained above the Percoll layer. Nonetheless, the detection of significant proportions (15-25%) of the total recovered activity of acid phosphatase and beta-galactosidase in the enriched chloragosome fraction supports the notion that some chloragosomes may be 'lysosome-like' organelles.
In the post-gizzard gut of the earthworm Lumbricus terrestris, distinguishing the functions of the luminal epithelium from those of the chloragogenous tissue has been hindered by the close apposition of these two tissues. Moreover, both tissues may have different functions from the anterior to the posterior of the animal. We analyzed the gut luminal contents of L. terrestris so as to gain a better understanding of the function of the luminal epithelium. The intestine was divided into four regions from anterior to posterior, and the water-soluble portion of the luminal contents of these four regions was analyzed for protease and amylase activity, calcium and ammonium ions, and protein. The same four regions of the gut wall were analyzed for glutamate dehydrogenase (GDH) and serine dehydratase (SDH) to determine their location with reference to the site of ammonia production. We observed high levels of proteases, amylase, protein and calcium ions in the gut luminal contents of the first two regions, and a significant decline of all four variables in region III. Conversely, ammonia was low in the gut contents of regions I and II but rose sharply in region III, which was also the region to which the tissue enzymes GDH and SDH were localized. The ammonia content of earthworm casts was observed to be much higher than that of the surrounding soil. These data are presented as partial evidence for the proposal that the excretory ammonia produced by feeding earthworms is a product of the luminal epithelium of region III of the gut. It is also proposed that ammonia and calcium may function as ion-exchangers in the absorptive function of the earthworm gut.
Morphometric analysis of transmission electron micrographs was used to compare the effects of metals on the multifunctional, metal-sequestering, chloragocyte cells of two epigeic earthworm species, Dendrodrilus rubidus and Lumbricus rubellus, inhabiting three field soils: a clean circumneutral reference soil (Dinas Powys); an acidic moderately Pb- and Zn-contaminated soil (Cwmystwyth); and a calcareous Cd-, Pb-, and Zn-contaminated soil (Draethen). The main findings were: (1) D. rubidus accumulated significantly higher tissue Cd and Pb and lower Zn concentrations than L. rubellus, especially at Draethen; (2) the volume fraction of chloragosomes was significantly lower and the volume fraction of debris vesicles significantly higher in D. rubidus from Draethen compared with L. rubellus at all sites and with the other two D. rubidus populations; (3) estimated relative toxicity factors, derived from soil metal concentrations and published EC50 data, suggested that the subcellular changes in chloragocytes, particularly in D. rubidus from Draethen, were caused mainly by Zn and Pb exposures; (4) scrutiny of the body burdens of each metal in both worm species across the three sites indicated that Cd was a major contributor to the structural changes observed in Draethen D. rubidus, and its impact was disproportionate to its soil and tissue concentrations in comparison with those of Pb and Zn. The apparent greater susceptibility of D. rubidus cells, compared with L. rubellus cells, to soil metal contaminants is discussed in light of differences in the quality and quantity of the metal body burdens accumulated by the two species. Further histopathalogic and morphometric studies on key organs and tissue of earthworms are required to provide biomarkers of exposure and to underpin linkage of biochemical-level changes and demography.
The effects of 48 h okadaic acid (OA) treatment and the ability to recover the induced toxicological injuries were examined in Enchytraeus crypticus of different ages (25 days and 3 months). The results demonstrated that the older worms are more sensitive to the toxin and show less capacity to recover. After 48 h OA treatment, the structural organization of the chloragogenous tissue appeared modified and associated with an immune response involving a higher number of circulating coelomocytes immunoreactive to anti-IL-6 antibody. The toxin effects were more evident in 3-month-old animals compared to specimens aged 25 days. Regarding the morpho-functional recovery from the induced modifications, first signs of recuperation were observed in younger worms at 48 h, and recovery was almost complete within 1 week. In older animals, the morphology of the chloragogenous tissue had not been restored, while a reduction in coelomocyte number was found after 1 week.
δ-Aminolevulinic acid dehydratase from Rhodopseudomonas spheroides, an enzyme which catalyzes the synthesis of the pyrrole, porphobilinogen, from 2 molecules of δ-aminolevulinic acid, has been purified 300- to 400-fold. The protein exhibits many of the characteristics of an allosteric enzyme. A plot of enzymatic activity versus substrate concentration yields a sigmoidal curve with an activity of about one-half that found in the presence of allosteric activators at high substrate concentrations. The allosteric effectors are monovalent cations, potassium, lithium, rubidium, and ammonium. A similar plot in the presence of these activators yields a hyperbolic saturation curve. Furthermore, the addition of K⁺ ions causes an association of the enzyme to form an equilibrium mixture of three enzymatically active species, a monomer, a dimer, and a trimer. The monomer, with a molecular weight of approximately 250,000, is the only species found on a sedimentation analysis in a sucrose gradient at very low concentrations of the above monovalent metallic cation. Sodium ions, on the other hand, do not fully activate the enzyme, as compared with the other alkali metal ions, whereas magnesium ions activate the enzyme to Vmax of about 80% of that found with K⁺, Li⁺, Rb⁺, and NH4⁺ ions. Manganese ions at low concentrations activate the enzyme but become inhibitory at higher concentrations.
The enzymatic activity is markedly inhibited by protoporphyrin and by hemin.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Aldrin epoxidase of the earthworm, Lumbricus terrestris L., has been shown to occur mainly in the intestine and seemed to increase in activity with the development of the animal. The microsomal fraction was identified by electron microscopy as the locus of the epoxidase. Although sesamex was not inhibitory, inhibition of expoxidase by carbon monoxide suggested the involvement of cytochrome P-450. However, the carbon monoxide difference spectrum of the microsomes was dominated by a material which was spectroscopically identical to earthworm hemoglobin.
1.
Details are given for the isolation of subcellular particles from the chloragogen tissue of the earthworm, Lumbricus terrestris. These particles exhibit succinic oxidase and cytochrome oxidase activity and are obviously identical with mitochondria.
2.
The protein of mitochondria amounts to 10–15% of total protein in chloragocytes.
3.
Mitochondria from the body wall were found to have higher succinic oxidase and eytochrome oxidase activity than those from chloragogen tissue.
4.
The influence of cofactor concentrations on oxidation of succinate was determined for the particles from body wall: the pH-optimum is near pH 7,4; the O2-consumption increases with rising concentrations of succinate and is stimulated by eytochrome c; addition of phosphate, ADP, ATP, and Mg++ is without effect on respiration.
1.1. A method for the purification of catalase from Lumbricus terrestris (Oligochaeta: Annelida) is reported.2.2. The steps are ammonia sulfate fractionation, acetone fractionation, calcium phosphate gel adsorption, DE-52 chromatography, crystallization.3.3. The crystals are needle-shaped and weakly birefringent.4.4. Lumbricus catalase has a mol. wt. of ca. 231,000 and consists of 4, probably identical, subunits of mol. wt. 57,700.5.5. The enzyme activity measured as Kat-f. was 52,400.6.6. The spectrum is a typical ferriheme-protein spectrum with maxima at 620, 540, 500, 405 and 280 nm. The ratio was 1.14, in accordance with the slightly smaller mol. wt. than bovine liver catalase.7.7. In most particulars the Lumbricus catalase is very similar to bovine liver catalase and other mammalian catalases.
1. The difference in arginase activity between the tissues of Eisenia and those of Lumbricus shows a relationship to the difference in urea output by living worms of the two species under the same dietary régime.
2. In Eisenia the difference in activity between the tissues of fasting and feeding worms is much smaller than in Lumbricus. The specific outputs of urea by living, fasting and feeding worms likewise differ less than in Lumbricus.
3. These facts strengthen previous evidence in favour of a Krebs-Henseleit type of mechanism for urea production in earthworms.
4. In Eisenia the difference in arginase activity between gut and body wall is similar to, but smaller than, that in Lumbricus, and the body wall makes a major contribution to the total activity.
5. The combined concentrations of ammonia-, amino-, and urea-nitrogen initially present in homogenates of the tissues of these worms are proportional to the combined amounts of the three components excreted per unit weight by living worms of the same species and régime.
6. The two species differ in a number of other properties investigated.
1.1. Cellular and intracellular localization of catalase and acid phosphomonoesterase in the midgut of Lumbricus terrestris was studied by use of tissue fractionation.2.2. At least 60–70% of the catalase resides in the chloragocyte cytosol and the remaining 30–40% resides in gut epithelium peroxisomes.3.3. One of the main functions of the chloragocyte catalase is probably scavenging for H2O2 arising from the interaction between blood heme-protein and oxygen.4.4. A simple method for the histochemical detection of cytosol catalase is proposed.5.5. About 10% of the gut acid phosphatase resides in chloragocyte lysosomes. The chloragosomes contain no acid phosphatase.
1.
Freie Porphyrine kommen bei Lumbricus terrestris L. im Hautmuskelschlauch als Protoporphyrin (350 58 g/g Trockengewicht) und im Mitteldarm als Koproporphyrin (41,4 1,5) und als Tricarboxyl-Porphyrin (7,9 0,5) vor. Die Koproporphyrinkonzentration ist hher im Chloragog (54,8 4,3) als in der Darmwand (34,8 1,4).
2.
Porphobilinogen ist weder in den Organen noch im Darminhalt oder im ausgeschiedenen Sand nachweisbar.
3.
Die Syntheserate von Porphyrinen betrgt bei 30 C in mitochondrienfreien Extrakten aus dem Mitteldarm 9,3 g/g Frichgewicht 4 h, aus der Darmwand 3,7, aus dem Chloragog 22 und aus dem Hautmuskelschlauch 1,12.
4.
Lumbricus scheidet keine freien Porphyrine aus; jedoch wurde in sehr geringer Menge eine Eisenprotoporphyrin-Verbindung identifiziert.
5.
Es wird die Verschiedenheit der Porphyrine bei Anneliden und allgemein bei Wirbellosen aus der unterschiedlichen Enzymausrstung erklrt, und es wird die Annahme begrndet, da das Hm des Hmoglobins bei Lumbricus im Chloragog synthetisiert wird.
1.
Die Gesamteisen-Konzentration betrgt hei Lumbricus terrestris 26018g/g Trockengewicht bzw. 423g/g Frischgewicht.
2.
Von dem Gesamteisen entfllt ungefhr je ein Drittel auf das Hmoglobineisen, auf das Fermenteisen und auf das Resteisen. Hahn (1937) hat beim Hund etwa gleich hohe Fermenteisen- und Resteisen-Konzentrationen gefunden.
3.
Von dem Resteisen sind 83,23,4% mit 0,8% Natriumchlorid-Lsung extrahierbar. Das extrahierte Resteisen ist nicht dialysierbar gegen diese Lsung.
4.
Die Gewebseisenkonzentration im Hautmuskelschlauch betrgt 1388g/g Trockengewicht, in den Nephridien 16810, in den Geschlechtsorganen 22112, im Chloragog 42743 und in der Darmwand 48558. Der Mitteldarm (nach Jordan 1913 Darmteil mit Chloragog, ber dessen Lnge sich die Typhlosolis erstreckt) enthlt 49343g/g Trockengewicht und der Restkrper (alle brigen Organe) 25814.
5.
Der Eisengehalt der Chloragosomen betrgt 84090g/g Trockengewicht. Auf diese Gebilde entfallen 472% des Eisens des Mitteldarmes und 804% des Chloragog-Eisens.
6.
Nach 4 Wochen dauernder Eisenftterung wurde die Eisenzunahme in verschiedenen Organen und Geweben gemessen. Lumbricus speichert Eisen im Zellplasma der Chloragocyten. Das maximale Speicherungsvermgen betrgt 1100–1300g/g Trockengewicht.
1. The properties of alkaline phosphatase in the nephridia of Lumbricus terrestris L. and the distribution in the nephridium and in the nephridia of various regions of the body have been determined. 2. The enzyme is activated by Mg++, shows pH-optimum at pH 9,6 and has Michaelis-constants of 1,3·10-3M/l β-glycerolphosphate and 0,42·10-3 M/l p-nitrophenylphosphate. The activities against β-glycerolphosphate and p-nitrophenylphosphate are as 1:3. 3. The specific activity of the whole nephridium is 0,9 μM β-glycerolphosphate or 3,7 μM p-nitrophenylphosphate/mg Protein·h. 4. The specific activity of the first to the second and the third lobe is as 7:4:1. In consequence of the different content of protein, there is no significant difference in the absolute activities in the three lobes. 5. The histochemical reaction is positive in the thin tube and the ciliated middle tube, weaker in the cells surrounding the tube in the first and second lobe. 6. The investigation of the activity in nephridia of various regions of the body yields an U-shape gradient with a minimum in the region of segments 37-60.
1.
Chloragog von einheimischen Lumbriciden wurde lichtmikroskopisch, elektronenmikroskopisch und histochemisch (Eisennachweis) untersucht.
2.
Chloragocyten enthalten bis zu einigen m groe Vakuolen mit dicker Einheitsmembran, in denen 120–220 groe Makromolekle eingeschlossen sind. Teilchenform und -gre lassen auf Hmoglobin schlieen. Es kristallisiert in den Vakuolen und zeigt dann hexagonale Muster oder Perioden von 80–220 .
3.
Eisen tritt als Ferritin auf im Grundcytoplasma, in Siderosomen und zusammen mit Hmoglobin in den Proteinvakuolen. Diese Verteilung des Speichereisens (Resteisen) wird mit den Verhltnissen in der Sugerleber verglichen. Auf den Zusammenhang mit dem Hmoglobinumsatz wird hingewiesen.
4.
Kleine und groe Chloragosomen sind von einer Hllmembran umschlossen. Kleine Chloragosomen tragen oft cytoplasmatische Anhnge, in denen Ferritin vorkommen kann. Die Chloragosomenkrper, nicht aber ihre Anhnge, lassen sich mit EDTA (pH 4,5) und verschiedenen Kontrastierungsflssigkeiten aus dem Schnitt entfernen. Die Chloragosomenkrper sind frei von Ferritin. Ihr Eisengehalt (Delkeskamp 1963, 1964) drfte nicht auf Ferritin beruhen.
5.
Die Chloragocyten adulter Tiere sind regelmig mit Mitochondrien ausgestattet, die ber 1 m lang und etwa 0,2 m dick sind. Ihre Matrix ist relativ dicht, die Flchenausdehnung der als Cristae ausgebildeten Innenmembran ist gering.
6.
Die Chloragocyten sind zur Durchschleusung von Makromoleklen befhigt (Pinocytose bzw. Sekretion). Richtung, Gre und Art des Stofftransportes bedrfen der Klrung.
7.
Zwischen den fortsatzreichen Chloragocyten erstreckt sich ein erweiterungsfhiges Labyrinth. In ihm findet man von der Zellmembran abstammende Blasen und andere Formbestandteile, die auch in den Chloragosomenhhlen vorkommen. Basale Fortstze liegen der Grenzmembran des Darmblutsinus breitflchig an, sind aber nicht wie die Muskelzellen mit Halbdesmosomen an ihr befestigt. Im basalen Bereich treten marklose Nerven mit Begleitzellen und sekrethaltige Zellen auf.
8.
Im brigen besitzen die Chloragocyten Zellkerne mit den gewohnten submikroskopischen Merkmalen und die bekannten Arbeitsstrukturen des Cytoplasmas wie Golgi-Apparat und Ergastoplasma.
1)
Chloragog tissue of native lumbricids was examined light- and electron microscopically as well as histochemically for its iron content.
2)
Chloragogcells contain vacuoles up to a few m in size which have a thick unit membrane; they contain macromolecules which are 120–220 in size. Judged by form and size these particles may be hemoglobin. It cristallizes in the vacuoles and shows hexagonal patterns or periods of 80–220 .
3)
Iron is found as ferritin in the cytoplasmic matrix, in siderosomes, and together with hemoglobin in the protein vacuoles. The distribution of storage-iron (residual iron) is compared with the situation in the liver of mammals. The relation to hemoglobin metabolism is pointed out.
4)
Small and large chloragosomes are covered with a sheath. Often small chloragosomes show cytoplasmic processes, which may contain ferritin. The bodies of chloragosomes, but not their processes, can be removed from the section by EDTA (pH 4.5) or certain electron stains. The bodies of chloragosomes are free of ferritin. Their iron content (Delkeskamp 1963, 1964) does not seem to be based on ferritin.
5)
In adult animals the chloragogcells always contain mitochondria, which are more than 1 m in length and about 0.2 m thick. Their matrix is relatively dense; the surface area of the inner membranes, forming the cristae, is small.
6)
Chloragogcells have a transport system for macromolecules (pinocytosis, secretion). Direction, magnitude, and nature of this transport system remain to be determined.
7)
The labyrinth between the numerous cell processes is able to enlarge. Vesicles derived from the plasma membrane and other particles are found in this labyrinth; the same kinds of particles are found in the chloragosome cavities. Basal processes cover the limiting membrane of the intestinal blood sinus; however, these processes are not attached to the membrane by semidesmosomes, as is the case in muscle cells. Non-myelinated nerves with satellite cells and cells containing secretion droplets are found in the basal region.
8)
Otherwise the chloragogcells have nuclei whith the usual submicroscopic structure and the well known organelles of the cytoplasm like Golgi complex and ergastoplasm.
1.
Es wird die Arbeitshypothese begrndet, da das Chloragog der Oligochten und die Mitteldarmdrsen der Crustaceen und Mollusken als Zentralorgane des Stoffwechsels analog der Wirbeltierleber komplexen Stoffwechselaufgaben auerhalb des allgemeinen Zellstoffwechsels dienen, insbesondere der Aufbereitung, Umformung und Bereitstellung von Nhr- und Baustoffen sowie der Synthese und dem Abbau krpereigener Verbindungen.
2.
Als erster Beitrag zur Verifizierung dieser Hypothese werden chemische Untersuchungen ber den Lipidbestand im Mitteldarm und dem brigen Krper (s. Tabelle 1) sowie ber Fettresorption und -speicherung bei Lumbricus terrestris L. vorgelegt.
3.
Der von Liebmann mit histologischen Methoden erhaltene Befund, da im Chloragog eine Speicherung von Neutralfett stattfindet, wird mit quantitativ-chemischer Methodik besttigt.
Der Mitteldarm kann maximal 5–7% des Frischgewichts an Neutralfett speichern. Da histologisch bewiesen ist, da die Speicherung berwiegend im Chloragog erfolgt, entspricht dieser Wert einem Lipidgehalt im Chloragog von 12–16% des Frischgewichts, das sind etwa 40–50% des Trockengewichts.
Die Speicherung ist in den verschiedenen Abschnitten des Mitteldarms verschieden, am grten in dem vordersten Abschnitt, der den strksten Chloragogberzug hat.
Etwa 4/5 des aufgenommenen Neutralfetts werden im Mitteldarm, 1/5 im brigen Krper gespeichert.
4.
Das im Chloragog gespeicherte Neutralfett wird im Hunger wieder abgebaut. Die so freigesetzte Energiemenge reicht vllig aus zur Deckung des Grundumsatzes.
5.
Die Resorptionsgeschwindigkeit fr Olivenl liegt mit 0,25 mg pro Tag und Quadratzentimeter resorbierender Oberflche in der gleichen Grenordnung wie bei den Sugetieren.
Die Ausnutzung gebotenen Olivenls betrgt etwa 12–25%.
6.
Bei niedriger Temperatur schmelzende Fettsureester werden von Lumbricus resorbiert, Glyzerinester (Olivenl, Triolein, -Monoolein) rascher als thylester (thylmyristat, thyloleat).
Hochschmelzende Glyzerinester (Tristearin) werden nicht aufgenommen, Caprylsureester haben Giftwirkung.
7.
Fettsuren und deren Salze (Myristin-, Palmitin-, Stearin- und lsure) werden von Lumbricus nicht resorbiert. Caprylsure, in geringem Mae auch lsure und ihr Natriumsalz sind giftig fr den Regenwurm.
8.
Es ist wahrscheinlich, da bei Lumbricus Fettsureester ohne vollstndige Hydrolyse resorbiert werden, mglicherweise in Trpfchenform.
1.(1) The chloragocytes of Lumbricus terrestris contain glycogen, which decreases during starvation, and two kinds of phospholipid.2.(2) The yellow-brown pigment could not be identified but some of its properties are described.3.(3) No trace of purine could be detected in the chloragosomes.4.(4) Minute particles of siliceous material are sometimes found in the chloragocytes.5.(5) The possible function of the chloragogenous tissue is discussed.
1.1. L. terrestris has no trehalose in the blood or coelomic fluid and is without trehalose activity.2.2. Glucose-6-phosphatase is also lacking and the concentration of glucose in the blood or coelomic fluid is very low (0.01–0.05 μg/μl normally).3.3. The body wall contains 80% of the lactate dehydrogenase activity of the worm and both the lactate formation and the reoxidation to pyruvate takes place in the body wall muscle itself.4.4. In accordance with this the glycogen-storing chloragocytes contain very little or no LDH.5.5. Thus the earthworm does not have the lactate-glucose exchange mechanism known from vertebrate liver and muscle and hormonal regulation of extracellular glucose is probably absent.6.6. Chloragocytes exhibit significant amylase- and maltase-activities, which may be responsible for glycogen break-down in the absence of the phosphorylase-glucose-6-phosphatase system.7.7. There are histological indications that chloragocytes, or their distal parts are released to the coelom to form trephocytes or eleocytes.8.8. They may then break up or be transported by the coelomic fluid to other parts of the body and their contents of glycogen and other materials released.9.9. Comparative and functional aspects of the above are discussed.
DOPA peroxidase activity of the cytosol of chloragocytes in the earthworm was proved by biochemical and histochemical methods, respectively. Chloragocyte DOPA peroxidase do not hydroxylate tyrosine, but it may oxidize besides DOPA other phenolie compounds in the presence of hydrogen peroxide. Probable participation of this enzyme in the detoxication of phenolie compounds and in the formation of chloragosomes was discussed.
The chloragog cells of the earthworm Lumbricus terrestris contain numerous granules, chloragosomes, which were analyzed for metals and phosphate by histochemistry, by use of an electron microscope X-ray microprobe (EMMA), and by chemical analysis of chloragosome preparations. Inorganic and organic phosphate each accounts for about 3% of the chloragosome dry mass, Ca for 2–3%, Zn for 1–3% and Mg for 0.2–0.4%. Carbonate is not present in chloragosomes. The average molar ratios Ca∶Mg∶Zn∶total PO4 are 1∶0.1∶0.3∶1. The Ca∶PO4 ratio is fairly constant (correlation coefficient 0.99), while the Zn∶PO4 ratio varies considerably. It is concluded that Ca is bound in the form of inorganic CaHPO4 and organic ROPO 3Ca (or possibly Ca-polyphosphate complexes). Mg may also be phosphate-bound, while Zn probably is not. Chemical analysis of the calciferous glands revealed a high concentration of Ca, small amounts of Mg and phosphate, but no Zn. It is concluded that Zn is not excreted through the calciferous gland.
Storage of Ca in the chloragocytes and excretion of CaCO3 by the calciferous gland may be physiologically linked. Regulation of the concentrations of Ca and HCO3−ions in the blood and coelomic fluid may assist in equalization of osmotic pressures during dehydration and rehydration. This regulation may be a major function of the chloragosomes.
The chloragosomes were discussed in relation to the “spherites” of various arthropods and molluscs and to the “cytosomes” of anoxia-tolerating molluscs.
The in vivo cumulation of neutral red, methylene blue and acriflavine in the body wall, midgut epithelium and chloragosomes of Lumbricus terrestris L. and Octolasium transpadanum Rosa, and the discharge of these dyes by chloragosomes was investigated by spectrophotometry. The chloragosomes cumulated the largest amount of the ingested dyes. Electron microscopy displayed the activation of the chloragogen cells and an increased chloragosome formation on the first days of dye intake. In dye-free environment, the dye content of the chloragosomes decreased rapidly. The above properties of the chloragogen cells indicate the role of these cells in the defence mechanism against the toxic effect of certain cationic xenobiotics ingested via the intestinal tract.
Iron was found at the highest concentration in the chloragogenous tissue with smaller amounts in the intestine and the lowest concentration in the body wall. Earthworms treated with Pb++ for 20 days showed a decrease in the concentration of Fe+++ in the intestinal tissue but no significant changes in the other tissue. The enzyme delta-aminolaevulinic acid dehydratase (ALAD) was not detected in isolated chloragosomes but it was found in the chloragocyte cytosol, body wall and intestine. L. terrestris treated with Pb++ showed a decrease in the activity of ALAD in all tissues where it was located and particularly in the chloragocyte cytosol.
A specific antibody elicited against NADPH-cytochrome c reductase inhibited cytochrome P-450- and NADPH-dependent hydroxylation of biphenyl by rodent liver and lung microsomal preparations. The inhibition profiles suggested that both the 2- and 4-hydroxylation of biphenyl were mediated by a common NADPH-cytochrome c reductase (NADPH-cytochrome P-450 reductase) and that the same flavoprotein species operated in liver and lung microsomes of corn oil- or 3-methylcholanthrene-pretreated rats and hamsters. An immunochemically identical NADPH-cytochrome c reductase also apparently functioned in the NADPH-supported metabolism of benzo(a)pyrene and ethylmorphine. NADH supported the microsomal metabolism of benzo(a)pyrene and ethylmorphine in liver and biphenyl in liver and lung, but the maximal rates of reaction were slower than when supported with NADPH. The Km of NADH for biphenyl 2- and 4-hydroxylations in control hamster liver microsomes were approximately 5 mm. Anti-NADPH-cytochrome c reductase globulin inhibited NADH-supported biphenyl 2- and 4-hydroxylase activities in corn oil- or 3-methylcholanthrene-pretreated rats and hamsters, even at NADH concentrations as low as 0.25 mm. These results indicate that the same flavoprotein reductase species which mediated NADPH-dependent biphenyl hydroxylase donated at least one electron for the NADH-supported hydroxylation.
Components of chloragosomes isolated from Octolasium transpadanum ROSA were separated by fractional extraction. The chemical composition of the fractions was determined and their effect on the anion- and cation-binding capability and on the lipophilic property of chloragosomes was studied. The acid-alcoholic extract of chloragosomes contained, among others, metalloporphyrins with 2 or 4 free -COOH groups; the residuum consisted of basic proteins. The metalloporphyrins and phosphatides gained by extraction with chloroform-methanol neutralize the basic groups of the chloragosome proteins, thus, an excess of free acidic groups develops, which is responsible for the polyanionic nature and cation exchange of chloragosomes. The apolar groups of phosphatides and carotenoids are responsible for the lipophilic nature of chloragosomes. Under experimental circumstances both in vivo and in vitro, organic cations were bound to chloragosomes, partially by a cation-exchange mechanism and partially by formation of more stable complexes. A small amount of anions may be absorbed by the bound cations. The diluted acidic extract of chloragosomes predominantly contain organic cations, in the present study riboflavin, flavin nucle0tide and thiamine were determined. Chloragosomes, owing to their structure, form complexes of varying stability with anions, cations and hydrophilic compounds equally. This feature explains their capability of cumulating trophic and toxic substances, while their redox activity is explained by their metalloporphyrin, flavin, thiamine and carotene content.
Measurements of the redox potential values in buffered salt solutions containing body wall homogenate, body wall homogenate with isolated chloragosomes and in both solutions enriched with NAD have shown that chloragosomes are specific electron acceptors which prevent the rapid decrease of the redox potential under anaerobic conditions. The substances responsible for the electron-acceptor activity are most probably identical with the flavins, carotenes and metalloporphyrins present in chloragosomes as shown by the visible absorption spectra of the extracts. The results support the assumption that chloragosomes may play an important role in the metabolism under hypoxic and anoxic conditions.
Acid hydrolases are not exclusively lysosomal enzymes as indicated by the specific case of beta-glucuronidase. This enzyme is a membrane protein found in the endoplasmic reticulum and in the lysosomes of the cell.
An earthworm saline commonly used in previous investigations of earthworm neuromuscular physiology has been shown to be inappropriate for such studies because the ionic composition of the saline does not correspond to that of earthworm body fluids. Problems encountered when using this saline include: increased excitability of motor axons innervating the longitudinal muscle, a decrease in the amplitude of external electrical responses of the muscle, an increased susceptibility of longitudinal muscle fibres to injury spiking, and a decrease in resting potentials of longitudinal muscle fibres (to a mean of – 36·1 mV). Such problems have been overcome by the development of a new physiological saline whose composition closely corresponds to the ionic composition of earthworm body fluids. In the new saline responses of the nerve-muscle preparation remain stable for more than 1 h. Resting potentials of longitudinal muscle fibres are also stable with time, the mean resting potential being – 47·9 mV.
The water-soluble yellow pigment of earthworm chloragogen-tissue is a flavin, probably riboblavin, conjugated with protein . The full significance of such a high concentration of this vitamin is far from clear.
1.1. The lipids of the chloragosomes of the earthworm Lumbricus terrestris L. contain cholesterol, phosphatidyl choline, phosphatidyl ethanolamine and an unknown complex lipid.2.2. Pigment associated with the lipids contains carotenoids.3.3. Accumulated evidence suggests that the odour components of chloragosomes are coumarins and that the yellow water-soluble pigments are hydroxy-coumarins and chromosomes (flavones and flavanols) derived from plant material ingested by the earthworm.
An EDTA titration method has been developed for the determination of calcium in small biological samples. Fresh or dried material is extracted with 1 N nitric acid and the extract is alkalinised to pH 13.4 and titrated with EDTA in the presence of Patton and Reeder's indicator and sodium tartrate. Barium, magnesium, manganese, iron, lead, zinc, oxalate, phosphate and sulphate do not interfere owing to the high pH and the presence of tartrate. Copper interferes, but up to 5 µg of copper can be masked with cyanide if the copper concentration does not exceed one tenth the calcium concentration. This method permits the quantification of calcium in the range 0.5–40 µg and with a reproducibility of ±2.5% or better.
1.1. NaHC14O3, ureido-C14-citrulline and 5-C14-ornithine were shown to be effective precursors of protein arginine in Lumbricus terrestris. C14 from NaHC14O3 and from ureido-C14-citrulline is incorporated into the guanidino-C, and from 5-C14-ornithine into the ornithine moiety of protein arginine. C14 from both NaHC14O3 and ureido-C14-citrulline is also incorporated into excretory urea.2.2. Lumbricus, which appears to normally be ammonotelic, becomes ureotelic when starved. There is a concomitant increased incorporation of bicarbonate-C14 into urea during this transition. In fed worms, citrulline formation is the limiting step in urea biosynthesis. During starvation, citrulline utilization becomes limiting.3.3. All five enzyme-catalyzed reactions of the urea cycle have now been demonstrated with cell-free preparations of Lumbricus gut tissue. The rates measured for these reactions in vitro will account for the total urea formed by whole worms. The increased formation of citrulline during starvation is due mainly to increased activity of ornithine transcarbamoylase as well as an increase in carbamoylphosphate synthetase activity.4.4. The earthworm thus possesses a complete urea cycle which may normally function to synthesize arginine. In the transition from ammonotelism to ureotelism during starvation, the cycle may then be utilized to detoxify the excess ammonia produced by increased protein catabolism.
Me/ho& of Enzymatic Analysis Beauchamp C. and Fridovich 1. (1971) Superoxide dis-mutase Arginine and urea biosynthesis in the earthworm Lumbricus terrestris
- H U Bergmeyer
Bergmeyer H. U. (1974) Me/ho& of Enzymatic Analysis. Academic Press. New York. Beauchamp C. and Fridovich 1. (1971) Superoxide dis-mutase. Analyt. Biochem. 44, 276287. Bishop S. H. and Campbell J. W. (1965) Arginine and urea biosynthesis in the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. 15, 51-71.
In defence of Haber-Weiss The nitrogenous
- G Cohen
Cohen G. (1977) " In defence of Haber-Weiss ". In Super-oxide and Superoxide Dismutases (Edited by Michelson A. M., McCord J. M. and Fridovich I.). Academic Press, London. Cohen S. and Lewis H. B. (1949) The nitrogenous
Über die Pigmente der Chloragosomen und ihre histochemischen Eigenschaften bei Lumbricus terrestris L
- Fischer
Superoxide dismutase
- Beauchamp
Etude radioautographique du rôle trophique des cellules ehloragogènes des vers de terre
- Valembois
Histochemische Untersuchungen über die metabolisehe Aktivitat der Chloragosomen von Lumbricus terrestris
- Fischer