The effect of lead incorporation on the elemental composition of earthworm (Annelida, Oligochaeta) chloragosome granules

The elemental compositions of chloragosome 'granules' in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.     

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes. An in vivo investigation using fully quantitative electron probe X-ray micro-analysis

The elemental compositions of chloragosome "granules" in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1 = uncharacterised; Pool 2 = P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the "control" chloragosomes. In Zn-contaminated chloragosomes, most (approximately 70%) was bound by P-containing ligand(s) but some (less than 1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.     

The effect of lead incorporation on the elemental composition of earthworm (Annelida,Oligochaeta) chloragosome granules

Summary The elemental compositions of chloragosome granules in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.     

Chloragosomes of lumbricidae as specific electron acceptors. In vitro investigations.

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.     

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthworm Eisenia foetida: cytochemical demonstration of acid phosphatase activity

Summary 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.     

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes

Summary The elemental compositions of chloragosome granules in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1=uncharacterised; Pool 2=P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the control chloragosomes. In Zn-contaminated chloragosomes, most (70%) was bound by P-containing ligand(s) but some (<1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.     

The lysosomes of earthworm chloragocytes: biochemical and morphological characterization

Nine latent and sedimentable acid hydrolases have been detected in the homogenates of earthworm chloragocytes. Their full activity was revealed by treatment with Triton X-100, a Waring blender treatment, freezing and thawing, hypotonic media or incubation at pH 5 and 25 degrees C. Solubilization paralleled the activation of the enzymes. Together with kinetic studies, these results indicate that the acid hydrolases of the chloragocytes are inside typical lysosome-like particles whose membrane is impermeable to their substrates. It could be shown by density equilibration centrifugation that the lysosomes of those cells constitute a heterogeneous population of subcellular particles distinct from the chloragosomes. Moreover, their digestive function has been directly demonstrated by the capture and degradation of serum albumin. The lysosomes of the chloragocytes have been clearly identified as polyvesicular bodies by electron microscopic analysis of the fractions obtained by density equilibration centrifugation and by examination of the whole tissue, as such or after endocytosis of serum albumin or ferritin. Finally, our results do not support a possible relationship between the lysosomes and the chloragosomes of the chloragocytes.     

The elemental composition of the chloragosomes of two earthworm species (Lumbricus terrestris and Allolobophora longa) determined by electron probe X-ray microanalysis of freeze-dired cryosections

Summary The elemental composition of the chloragosomes of the two earthworm species, one with complex highly active Ca-secreting calciferous glands (L. terrestris) and the other with non-secretory glands (A. longa), was determined by the electron probe X-ray microanalysis of unfixed freeze-dried cryosections. The predominant constituents of the chloragosomes of both species were P, Ca, Zn and S, with lesser quantities of K, Cl and Fe. The most striking species differences in chloragosomal chemistry were the higher concentrations (expressed as relative mass fractions) of P(×2.1), Ca(×1.5), and Zn(×2.3) in L. terrestris, and the much higher S(×19.6) in A. longa. These differences were discussed in relation to the general ecophysiology of the two species, and more specifically in relation to heavy metal uptake and binding.     

The accumulation and intracellular compartmentation of cadmium,lead, zinc and calcium in two earthworm species (Dendrobaena rubida and Lumbricus rubellus) living in highly contaminated soil

Summary The earthworm Lumbricus rubellus contained more Ca and Zn, and less Pb and Cd, than Dendrobaena rubida living in the same contaminated disused-mine soil. Differences in the kinetics of Ca turnover may account for some of the inter-specific differences in heavy metal burdens, although the calciferous glands do not seem to be directly involved in heavy metal excretion. A comparison of the present findings with published data indicated that the concentration of soil Ca and the bioavailability of heavy metals, both factors being allied to soil pH, are important exogenous determinants of heavy metal accumulation by different earthworm populations. Electron microprobe X-ray analysis of air-dried smears of chloragogenous tissue showed that the metals were fairly specifically compartmentalized into two distinct organelles in both worms: Ca, Pb and Zn were found (associated with P) in the chloragosomes; Cd was found (with S and probably in stoichiometric association) in a more electron-lucent vesicular component, designated the cadmosome, but which may be identical with the debris vesicles which are characteristic inclusions in conventionally-fixed chloragocytes. The in vivo incorporation of Pb by the chloragosomes of D. rubida was accompanied by the loss of Ca, Zn and P.     

Structural basis of cation exchange, complex formation and redox properties in chloragosomes.

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.     

The effect of carbofuran-toxication on the chloragogen tissue of Tubifex tubifex Müll. (Oligochaeta).

Chloragogen cells, subserving ion exchange and electron accepting functions, were studied in Tubifex tubifex after insecticide treatment. Chloragogen cells were strongly influenced by in vivo carbofuran poisoning. The first alterations in the chloragogen cells became activated, both the formation and release of the chloragosomes reached a high rate. The released chloragosomes were phagocytosed by the amoebocytes. At an advanced stage of the toxication a heavy loading of the apical cytoplasm of chloragogen cells with lipid droplets, finally degenerative changes both in the chloragogen cells and amoebocytes were observed. Possible mechanisms of the carbofuran toxication and of the protective function of chloragogen cells in T. tubifex are discussed.     

Cold-stress induced formation of calcium and phosphorous rich chloragocyte granules (chloragosomes) in the earthworm Eisenia fetida

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.     

The effect of sublethal lead exposure on the ultrastructure and on the distribution of acid phosphatase activity in chloragocytes of earthworms (Annelida, Oligochaeta)

Summary Laboratory experiments were conducted to study the effects of the exposure to a sublethal concentration (500 p.p.m.) of lead on the ultrastructure and acid phosphatase compartmentalization of the chloragogenous tissue of earthworms,Eisenia foetida. For the cytochemical demonstration of acid phosphatase activity, lead and cerium were used as capturing agents. In both cases there was a change in the compartmentalization of acid phosphatase, the enzyme activity being localized within the chloragosomes in controls, but distributed throughout the cytosol in treated animals. In addition, acid phosphatase activity increased following lead exposure. At the ultrastructural level, disruption of the chloragosomal membranes, an increase in chloragosomal fusion processes and vesiculation of the cytoplasm were evident. Moreover, an enhanced release of chloragosomes to the extracellular space was found in lead-exposed worms.     

In vivo cumulation and discharge of azine, thiazine and xanthene dyes and their effects on the chloragogen cells of Lumbricidae (Oligochaeta)

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.     

Uptake and long-time storage of natural and synthetic dyes by earthworm chloragocytes. In vivo and in vitro investigations

Cationic or anionic dyes adsorbed onto cellulose granulate were transported across the gut wall, bound to blood proteins, and accumulated by the chloragocytes. Solubility in water promoted accumulation. The dyes ended up mainly in the chloragosomes. Down to 20 μmol dye per litre soil water resulted in visible accumulation. Worms which after dye-exposure were kept dye-free for 5 months retained substantial amounts of dye in the chloragosomes. In vitro experiments indicate that the binding to chloragosomes of synthetic and natural phenolics is by ion exchange with calcium phosphate and with an uncharacterized matrix-bound calcium chelator, aided by hydrophobic interactions between the dye and constituents of the chloragosome matrix. The findings are relevant for the evaluation of the effects of constant or periodic soil contamination with industrial or agricultural organochemicals.     

Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.     

The occurrence and localisation of heavy metals and glycogen in the earthwormsLumbricus rubellus andDendrobaena rubida from a heavy metal site

Summary The lead content of whole earthworms, highest in contaminated site specimens, was significantly higher inDendrobaena rubida thanLubricus rubellus and a species difference in zinc was also recorded. Selective feeding and differential absorption are discounted and a species difference in maximum tolerance to body lead is suggested. Copper was low in all specimens. Chloragocytes and intestinal tissue showed significantly higher lead levels in contaminated earthworms than in control material. Ultrastructurally, chlorgocytes from contaminated earthworms showed electron dense flecks associated with the chroragosome peripheries and within the debis vesicles. Very fine flecks occurred in the nuclei, but mitochondria and Golgi were indistinguishable from control material. Preliminary X-ray microanalysis of contaminated chloragocytes revealed lead and phosphorus. The deposits within the chloragocytes might represent unbound lead precipitated by phosphate buffer; flecks being absent from contaminated, citrate buffered material and from control material. The chloragosomes are proposed as possible sites for sequestered lead.Chloragocyte and intestinal glycogen levels were significantly higher in control material where the chloragocyte cytoplasm was rich in -glycogen rosettes, these being absent from lead contaminated cells. The glycogen-lead correlation suggests that the metabolism of contaminated chloragocytes is directed towards lead sequestration, though differing nutritional states cannot be ignored.The assistance of Mr. P.M. Webster, University of Keele, is gratefully acknowledged.     

A morphological and electron-microprobe study of the inorganic composition of the mineralized secretory products of the calciferous gland and chloragogenous tissue of the earthworm,Lumbricus terrestris L.

Summary The two pairs of lobes of the calciferous gland of the earthworm Lumbricus terrestris are specialized oesophageal diverticulae that secrete spherites ranging from 0.5–7.0 m in diameter. Correlative transmission and scanning electron microscopy indicated that the spherites (which are predominantly CaCO₃) are formed extracellularly in distinctive bays bounded by secretory-cell processes, and are mobilized anteriorly from the gland lumina to the lumen of a non-secretory pouch, where the majority coalesce and undergo phase transformation to concretions 0.5–1.0 mm in diameter consisting of a mass of cuboidal crystals with facets up to 40 m. The distribution of Sr (0.1 ml 5 % SrCl₂ injected into the posterior coelomic cavity) was monitored in the mineralized secretory products of the calciferous glands by X-ray microanalysis of 10 m — thick air-dried cryostat sections in a SEM. Strontium was not detected in chloragosomes at 2 h and 24 h post-injection. Strontium was transported anteriorly and specifically incorporated into gland spherites (detectable within 2h). This technique of Sr localization afforded sufficient structural and analytical resolutions to provide a confirmation of the sequence of extracellular changes in the gland/pouch system. In addition we were able to distinguish a population of growing spherites from the vast majority of mature spherites; size alone was a singularly poor indication of spherite growth.The major element constituents of the chloragosomes were P, Ca and Zn (Ca: P ranging from 0.4 to 1.0; Zn: P from about 0.05 to 0.45). Analysis of individual spherites showed that Ca was probably bound to P or P-containing matrix components, whilst Zn was probably linked to one or more different but unknown constituents.     

Scanning electron-microscopic study of the involvement of coelomic cells in earthworm antibacterial defense

Summary The first steps of an antibacterial reaction in the earthworm Eisenia fetida andrei were investigated. The main cellular mediators of this activity are the chloragocytes, a class of free coelomocytes existing only in annelids. Our observations using scanning electron microscopy have shown that chloragocytes were able to agglutinate and perhaps to destroy pathogenic bacteria such as Bacillus megaterium in the same way that they agglutinate and lyse vertebrate erythrocytes. Bacteria known to be non-pathogenic for the worm, such as Acinetobacter, were not agglutinated but slowly eliminated by segregation into brown bodies. Chloragocytes maintained in vitro, lost their chloragosomes and exhibited stronger agglutination activity against pathogenic bacteria than chloragocytes in situ. From this increased efficiency of chloragocytes in vitro, we infer that, in normal living conditions, chloragocytes probably intervene in antibacterial defense mainly after their extrusion from the coelomic cavity and their spreading and degranulation at the surface of the integument.