Earthworm coelomocytes as a soil health assessment tool

Different pollutants can disrupt earthworm coelomocytes integrity and functions, and their responses can be applied as biomarkers of sublethal contaminant exposure. In this context, the aim of this study was to develop an in vitro protocol for coelomocyte extraction, maintenance and analysis with regard to soil health status and chemical toxicity profile assessments. The extrusion technique was first tested comparing previously depurated (purged stomach content) and non-depurated and resampled earthworms. After testing, earthworms were exposed to different 2,4D and chloroacetamide concentrations for methodology validation. The values of viability were not affected by food restriction since no statistical difference was observed between non-depurated (sample A) and depurated (sample B) organisms. Regarding to cell density, a significant (p<0;05) reduction of 22% was observed between non-depurated and depurated organisms, indicating that food restriction may affect cell density. However, the non-depurated resampling did not show a significant reduction, indicating that this assessment may not be affect by resampling of the same organism. For both chemical compounds, no change in cell viability was observed at all assessed concentrations and exposure times. However, for cell density, a mainly time-dependent effect was observed for organisms exposed to chloroacetamide, and concentration-dependent effect for organisms exposed to 2,4D. The proportion of immune system cells was altered, mainly after 24 h, with the increasing of granular amoebocytes proportion. The difference in the proportion of granular amoebocytes in earthworms exposed to 2,4D can be explained by the existence of recognition and elimination mechanisms for this chemical substance. Thus, assessments of pollutant responses with in vitro coelomocytes seem to be a powerful tool for ecotoxicological studies.

Metabolism of zinc in the mussel, Mytilus edulis (L.): a combined ultrastructural and biochemical study

The uptake, transport, storage and excretion of zinc has been studied in Mytilus edulis. Zinc accumulates in the soft tissues in proportion to its concentration in sea water whilst the concentration in the haemolymph is little above that in the environment. Uptake is via the gut, mantle and gills. The zinc is transported from the gills and gut (t½ ≈ 8 days) via the haemolymph, either as a high molecular weight complex or in the granular amoebocytes, to the kidney. Most of the body zinc is present in the granular amoebocytes (which are found in all the body tissues) or in the gut and kidney. The kidney forms the major storage organ for many trace metals, containing 30% of the body zinc and a concentration of about 1000 μg/g. Zinc is localized as insoluble granules in membranelimited vesicles occupying some 20% of the cell volume. Excretion of zinc is by defaecation, exocytosis of the kidney granules into the urine and diapedesis of the amoebocytes. A multicompartmental model for zinc metabolism which correlates the ultrastructural and kinetic data is proposed.

Memoirs: Studies on the Cultivation of Pieces of the Mantle of Modiolus Modiolus

1. Pieces of the mantle of Modiolus were sterilized by means of ultra-violet light, and cultivated, by the hanging drop method, in sea-water, and in sea-water plus tissue extract. 2. An outwandering of amoebocytes takes place shortly after the preparations are made; this is followed by an outgrowth from the epithelium which secretes the shell. Three types of epithelial cells are present in the outgrowths. 3. Undoubted stages of mitosis or of amitosis were not observed amongst the epithelial cells. 4. The shell epithelium often becomes folded so that hollows are present on the surface of the explant. The cells at the margin of the folds become elongate and tend to grow over the hollows. 5. The amoebocytes, by means of membranous expansions of the ectoplasm and fine pseudopodial-like processes, undergo movements and change of shape. Clumped and necrotic cells are rounded. 6. Hyaline and finely granular amoebocytes, due to their phagocytic action, become filled with granules, vacuoles, and large deeply stained bodies. 7. The amoebocytes often form a loose network in the medium. 8. Stages of mitosis or of amitosis were not observed, but amoebocytes with double nuclei were present in some of the preparations.

Memoirs: On the Nature and Functions of the Amoebocytes of Ostrea edulis

1. There are two kinds of corpuscles in the blood of the oyster; one consists of granular, the other of hyaline amoebocytes. 2. The granular amoebocytes are amoeboid, though the speed of their movement is slow. 3. The granules are yellow or yellowish green in the fresh condition. 4. The granules are neutrophil with a tendency to become stained by the basic dyes intra vitam. 5. Granules can never be distinguished in fixed and stained amoebocytes. 6. The amoebocytes are distributed everywhere throughout the body, an especially large number being normally present around the gut. 7. The so-called ‘pseudopodia’ of the amoebocytes are described and their nature discussed. 8. The effects of several kinds of reagents on amoeboid movement and on the amoebocytes are described. 9. The amoebocytes become entangled with one another by bristle-like ‘pseudopodia’, or by elongated strands of hyaline ectoplasm outside the body. There is no true coagulation of the blood, and no fibrin production. 10. The mechanism of phagocytosis is described and discussed. 11. Sucroclastic, lipoclastic, and proteoclastic enzymes are present in the amoebocytes and enable the amoebocytes to digest intracelluarly. 12. The optimum action of the amylase is about pH 7.0 and of the proteoclastic enzymes about pH 8.0. These optima are not very well marked. 13. A complete oxidase system is present, revealed by the indophenol reaction, by the power of reducing indigo carmine, a process which is irreversible, and by a slight reaction with guaiacum. 14. The amoebocytes can absorb glucose both in the gut and the mantle cavity. 15. There is no evidence of absorption of soluble matter nor of solid substances by the epithelium of the mantle cavity, e.g. gills, labial palps, &c, other than by the agency of amoebocytes. 16. The amoebocytes play a prominent part in excretion. They reject directly foreign or indigestible matter by way of the epithelium of the excretory organ, pericardium, surface of the auricle, rectum, and mantle cavity.

Memoirs: On Merlia Normani, a Sponge with a Siliceous and Calcareous Skeleton

Merlia is a vermilion coloured incrusting Monaxonellid sponge belonging to a new sub-family--Merlinas--of the Haploscleridæ. Large granular amœbocytes (calcocytes) have constructed a basal calcareous skeleton formed of vertical tubes divided up by horizontal tabulte. The tubes are built up of columns, each with three vertical wings which unite with wings of neighbouring columns to form tubes. This mode of construction was probably primarily determined by the disposition of the branches of the choanosome which led to the deposition of amœbocytes at the points of bifurcation of the lines of flagellated chambers. Apparently the calcocytes become wholly transformed into lumps, conules or flakes. The calcareous skeleton shows certain resemblances, especially at the surface, to certain Palceozoio fossils, classed among " Tabulate corals " or Polyzoa. There is no dermal epithelium, and the canal system is hymenopylous. The sponge has been found in 60-90 fathoms off Porto Santo Island and Madeira. A few more words remain to be said. It has been denied that Merlia is a spouge. I can only say that my opinion whether riglit or wrong, is based on the prolonged investigation of abundance of good material, whereas other opinions seem to me to be founded mainly on à priori considerations. I have examined over 500 specimens of Merlia and have always found the tissues of the sponge in most intimate association1 with a calcareous structure, which grows as the sponge grows. Granular amœbocytes varying in shape according to circumstances are found everywhere in contact with the skeleton, and apparently in continuity with the organic matrix of the same. Also there is a curious similarity in appearance between the granules of these superficially placed cells and the surface view of the ends of the fibrillte of the skeleton. The amœbocytes in the upper part of the sponge have the same fundamental characters as those in the interior of the crypts. Amœbocytes are found deep down in crypts nearly closed over by tabulte, and it is incredible that these large masses of cells could have worked their way down through the almost closed slits which are found in many tabulæ. Apart from these mechanical difficulties, one cannot imagine, from the point of view of common-sense, why the under surface of a supposed parasitic sponge should send down cylindrical moniliform masses of granular cells into the empty spaces of a foreign organism, thereby carrying out a seemingly useless and exhausting procedure. In young sponges on delicate shells, well stained and perfectly transparent, it can be clearly seen that there is not the least trace of any other organism than the sponge (on the shell). Weltuer writes--and not unnaturally--of the calcareous structure as that of an unknown organism in which a sponge has settled. If this be so the said organism has preserved its incognito in a marvellous manner. The theory that Merlia is a sponge that lias formed both a siliceous and a calcareous skeleton seems to me the only one possible, and further, the theory that it is a siliceous sponge that has taken to forming carbonate of lime one that is extremely probable.