Coelomic fluid

Wampler and Jamieson (1980) studied 12 species of luminous earthworms belonging to six genera (Diplocardia, Diplotrema, Fletcherodrilus, Octochaetus, Pontodrilus and Spenceriella) from the United States, Australia and New Zealand, and found that all of the species exude luminous coelomic fluid from their dorsal pores, except Pontodrilus bermudensis that exudes the fluid from the mouth. All of them emit luminescence of broad emission spectra with the peaks ranging from 500 nm to over 570 nm. 

Extraction and purification of Diplocardia luciferase (Bellisario et al., 1972). About 50 specimens of Diplocardia longa (widespread in southern Georgia about 30 cm in length) were electrically stimulated in 250 ml of 0.1 M EDTA at 4°C to exude coelomic fluid. The suspension of coelomic cells obtained was centrifuged at 480 g for 5 min. The pellets from 200 worms were combined and an acetone powder was prepared. The acetone powder obtained (about 10 g) was stable at -80°C for at least one year. 

Goven, A.J., S.C. Chen, L.C. Fitzpatrick, and B.J. Venables. 1994. Lysozyme activity in earthworm (Lumbricus terrestris) coelomic fluid and coelomocytes enzyme assay for immunotoxicity of xenobiotics. Environ. Toxicol. Chem. 13 607-613. 

Hemerythrin is a respiratory protein isolated from sipun-culids (marine worms). All sipunculids examined have, in the coelomic fluid, erythrocytes loaded with the protein which in most species so far examined is octameric, but sometimes tri-meric (18, 19) and in one instance dimeric and tetrameric (20, 21). From the retractor muscle of Themiste zostericola, the protein has been characterized as a monomer (22). The monomer (23) and the subunits of the trimer (24) and octamer (25) are remarkably similar in tertiary structure, having a M.W. of about 13,500 daltons. Each subunit contains one binuclear iron site. There is no porphyrin ring and the irons are coordinated only to amino acids, some of which, as well as probably an oxy group, form the binding atoms (26). 

For the metabolism rangefinding experiments, sea urchins were dosed by injection at a level of 2.2 mg/kg of body weight with -PNA into the central cavity, held in untreated water at 12°C, and both the water and coelomic fluid sampled periodically over the course of 8 hours. Most of the PNA was eliminated rapidly into the water (Fig. 2), with a pseudo-first order half-life of appearance of 12 minutes half of the test compound was lost from the coelomic fluid in about 24 minutes. 

Figure 2. Excretion rate of PNA from S. purpuratus (H), radioactivity in the water (X, O), radioactivity in coelomic fluid (2 replicates).

Motokawa, T. (1982). Rapid change in mechanical properties of echinoderm connective tissues caused by coelomic fluid. Comp. Biochem. Physiol. 73C, 223-229. 

Bartels-Hardege, H.D., Hardege, J.D., Zeeck, E., Muller, C., Wu, B.L., and Zhu, M.Y., Sex pheromones in marine polychaetes V a biologically active volatile compound from the coelomic fluid of a female Nereis (Neanthes) japonica (Annelida Polychaeta), J. Exp. Mar. Biol. Ecol., 201, 275, 1996. 

When alarmed, earthworms secrete coelomic fluid through dorsal pores, located in the grooves between the segments. To trigger release of the alarm secretion, one can shock an earthworm electrically (obviously not exactly a natural stimulus) with current from two size D batteries while on a wax paper. The worm will secrete coelomic fluid. A 2-cm piece of this paper with secretion can be cut out and presented to another, naive earthworm. Observe the response rearing up and withdrawing. Compare this behavior with the response to control stimuli such as salt solution or the normal mucus from an undisturbed earthworm (Rosenkoetter and Boice 1975). 

Earthworms can be purchased from a bait dealer. Use Eiseniafoetida or Diplocardia riparia. (Lumbricus terrestris responds positively to coelomic fluid, a behavior which is harder to recognize.) Keep the worms in a bucket in peat moss, old leaves, or commercial earthworm bedding. Worms must be healthy and cahn, i.e., crawhng slowly forward, do not jerk about rapidly, and do not tend to crawl backwards or defecate. 

Coelomic fluid fluid of the coelom, the main body cavity in which the gut is suspended, in many animals having a body made up of three parts (ectoderm, mesoderm and endoderm). 

Bundy, J.G., Osbom, D., Week, J.M., Lindon, J.C. and Nicholson, J.K. (2001) NMR-based metabonomic approach to the investigation of coelomic fluid biochemistry in earthworms... 

Bundy, J.G. Osborn, D. Weeks, J.M. Lindon, J.C. Nicholson, J.K. An NMR-based Metabonomic Approach to the Investigation of Coelomic Fluid Biochemistry in Earthworms under Toxic Stress, FEES Fetters 500(1-2), 31-35 (2001). 

The story of olean (116) was given already. Another example is 5-methyl-3-heptanone (125), which is the pheromone in the coelomic fluid of gravid specimens of Nereid marine polychaetes (Platynereis dumerilii). It is responsible for the induction of the nuptial dance behavior prior to the release of gametes in P. dumerilii. The female-produced (.S )-125 attracts the males, while the male-produced (R)-125 is active on females.147,148... 

NMR studies performed on tissue extracts on the coelomic fluid of Eisenia andrei and Eisenia fetida allowed to distinguish the two species one from each otlier (Bmidy et al. 2002). We have seen in all over this book that fluorescence spectroscopy can detect differences that occur on the structural and dynamical levels purified macromolecules. The following data will show that it is possible to use fluorescence spectroscopy to detect differences in the cells at the structural and the metabolical levels. 

Figure 10.2. Fluorescence emission spectra of coelomic fluid of Esenia fetida (b) and Esenia andrei (a) obtained at ex = 320 nm. 370 nm. Temperature = 20°C and pH 8.6.

The fluorescence excitation spectrum of a fluorophore characterizes the electron distribution of the molecule in the ground state. Thus, it helps to identify the structure and / or the nature of the emitting molecule. The fluorescence excitation spectrum recorded on the coelomic fluid of Eisenia andrei shows that the fluorophore absorbs at a peak equal to 314 nm at pH 8 (Fig. 10.3). Fluorescence properties of the emitting fluorophore are characteristic of the 4-methylumbelliferyl p-D-glucoronide (MUGlcU) (Molecular Probes, 1992-1994). The absence of fluorescence in Eisenia fetida indicates that the metabolism of this substrate is inhibited by one or many enzymes present only in the coelomic fluid of Eisenia fetida. 

Excitation at 280 nm of the coelomic fluid yields a fluorescence emission at 333 nm characteristic of dissolved protein (data not shown). Addition of 2-p-toluidinyl-naphthalene-6-sulfonate (TNS) induces a decrease in the fluorescence emission of the proteins and an increase of TNS fluorescence indicating an interaction between the extrinsic fluorophore and the proteins. 

In presence of coelomic fluid, FNS shows a significant fluorescence. However, the position of the peak varies with the origin of the coelomic fluid. In fact, the emission peak of TNS is equal to 397 nm and 410 nm for Eisenia andrei and Eisenia fetida, respectively (Fig. 10.4). This difference is the result of the contribution of the emission of the coelomic fluid to the recorded emission spectrum. In fact, since there is no fluorescence occurring from the coelomic fluid of Eisenia fetida., the observed emission in presence of TNS is from the extrinsic fluorophore only. Meanwhile, the spectrum recorded for Eisenia andrei is the result of the contribution of both TNS and the natural fluorophore present in the coelomic fluid. Experiments with TNS confirm the fact that coelomic fluids of Eisenia andrei and Eisenia fetida differ, i.e., the metabolism of the two earthworms is not the same. 

Figure 10.3. Fluorescence excitation spectrum of coelomic fluid of Eisenia andrei. nm. The peak of the spectrum is located at 314 nm. Temperature = 20°C and pH = 8...

Figure 10.4. Normalized fluorescence emission spectra of coelomic fluid of Eisenia andrei = 370 nm) (a), of TNS bound to Esenia andrei 397 nm (b) and of TNS bound to Esenia fetida 410 nm (c). Xex 320 nm. Temperature 20°C and pH = 8.

In conclusion, fluorescence data shown indicates clearly that Eisenia andrei and Eisenia fetida display different molecular composition in their coelomic fluid and thus have different metabolisms. This work reveals for the first time the possibility of using fluorescence spectroscopy as a tool to perform taxonomic studies. 

Table 1. Lipid concentrations of cell-free coelomic fluid or hemolymph from selected marine invertebrates...

Clark, M. E. (1964). Biochemical studies on the coelomic fluid of Nephtys hombergi (Poly-chaeta Nephtyidae), with observations on changes during different physiological states. Biol. Bull. 127 63-84. 

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