Introduction

The body fluid filled in the space between body wall and gut is called coelomic fluid (CF). The coelomic fluid of earthworms contain various molecules which exhibit important biological properties such as antimicrobial substances^1-4,  hemagglutinating⁵ and hemolytic agents⁶.  The primary function of the CF presumably, is to destroy membranes of foreign cells, a mechanism that causes cell death by cytosol release and is attributed to coeolomocytes which secrete humoral effectors into the coelomic fluid. The components of the CF are lectin in character^7-9.

The present study reveals the comparative characterization of CFs of Eisenia foetida and Perionyx excavatus with respect to their effect on coagulation and hemolysis.

Materials and Methods

Perionyx excavatus and Eisenia foetida were obtained from GKVK (Gandhi Krishi Vignyan Kendra) Bangalore, and were cultured. Uniplastin kit was purchased from Tulip Diagnostics (P) Ltd. Goa. Human blood samples were from healthy volunteers. All other chemicals were of analytical grade purchased from S.D. fine chemicals and HiMedia laboratories, Mumbai, India.

Culturing of Earthworms

Eisenia foetida and Perionyx excavatus were cultured on suitable bedding in plastic trays.  Under ideal conditions, they were fed with organic substances from plant and animal origin. The feed stock chosen were dairy and beef manures which is considered as best natural food for the Earthworms¹⁰. 75%- 80% moisture content were maintained so that the average worm weight and the reproduction rate increased¹¹. As the worms are aerobic, sufficient aeration was provided.

Extraction of Coelomic fluid

Extraction of Coelomic fluid from chosen earthworm species was performed according to the method of Kale R., (1991)¹². The earthworms were washed with cold water 3-4 times at room temperature and their body surface were dried on filter paper.  Then they were subjected to heat shock. Heat shock was performed by using 35°C – 40°C water in suitable container which was rubbed against the body of the earthworms. The procedure is repeated for 3- 4 times. The coelomic fluid obtained from the body of the earthworms was then collected and stored in vials.

Estimation of protein

Protein was measured by the method of Lowry et al., (1951)¹³ using bovine serum albumin  standard (0-75µg).

Assay of Hemolytic activity

Preparation of washed erythrocytes

Human blood was collected from the vein of healthy volunteers who had not taken any medication for at least 2 weeks and were non-smokers. 9 volumes of blood were collected into 1 volume of acid citrate dextrose. Centrifugation was performed at 5000rpm for 10min. Supernatant was discarded and the pellet (packed erythrocytes) was washed in phosphate buffered saline (PBS) two times and used for the assay.

b) Assay of hemolytic activity

To assay direct hemolytic activity, the washed erythrocytes were suspended in 9 volumes of PBS and 1ml of suspension was incubated separately with  60µg/ml of CF from Eisenia foetida and  105µg/ml of CF from Perionyx excavatus at 37°C for 30min. The reaction was stopped by adding 10ml of ice cold PBS and centrifuged at 4°C for 10min at 800rpm. The amount of hemoglobin released in the supernatant was measured at 540nm. The experiment was repeated three times. For control, to 1ml of packed erythrocytes 9 volumes of water was added and the result was accounted as 100% hemolysis¹⁴.

Coagulation Assay

a) Preparation of plasma

Human blood was collected from the vein of healthy volunteers who had not taken any medication for at least 2 weeks and were non-smokers. 9 volumes of blood were collected into 1 volume of acid citrate dextrose. Centrifugation was performed at 5000rpm for 10min. The supernatant collected was plasma.

b) Assay of Prothrombin time

Assay for coagulation is based on prothrombin time. For concentration dependent assay, different concentrations (10µg/ml – 60µg/ml) of protein sample was added to 0.1ml of plasma. Incubation was done for 10min. 0.2ml of prothrombin reagent was added after the incubation. The time taken to form a clot in each case was recorded.  For time dependent assay, the clot formation was recorded using a protein concentration of 40µg/ml at different time intervals (2min – 30 min).  The experiment was repeated three times.

Results and Discussions

The CF of Perionyx excavatus exhibited hemolytic activity on washed intact erythrocytes. The activity was concentration dependent whereas CF of Eisenia foetida did not exhibit any hemolytic activity on washed erythrocytes. This result is in constrast with the result obtained from earlier workers⁶ (Sven Lange et al., 1999). This may be due to geographical variations in the contents of CFs from the same species. The results revealed the absence of hemolytic factors responsible for the hemolytic activity in the CF of Eisenia foetida and their presence in CF of Perionyx excavatus. The hemolytic activity of CF of Perionyx excavatus was concentration dependent as shown in the fig. 1.

Figure 1: Hemolytic activity of CF of Perionyx excavatus.   Click here to View figure

(Each value represents mean of three independent determinations)

Coagulation assay was performed to the CFs of Eisenia foetida and Perionyx excavatus. Both the coelomic fluids were found to be procoagulants. The prothrombin time was dependant on concentration of protein (fig.2 and fig.3) and incubation time (fig.4 and fig.5). The results clearly indicate that with the increase in concentration of the protein, the time taken to form clot decreased clearly indicating the procoagulant activity.

Figure 2: Prothrombin time of CF of Eisenia foetida.   Click here to View figure

(Each value represents mean of three independent determinations)

Figure 3 : Prothrombin time of CF of  Perionyx excavatus.   Click here to View figure

(Each value represents mean of three independent determinations)

With 40µg/ml protein in CF of Eisenia foetida and 80µg/ml protein in CF of Perionyx excavatus,  prothrombin time was recorded at different incubation time. An increase in the incubation time resulted in a decrease in the clotting time (fig.4 and fig.5).  The results indicate that the procoagulant potency of CF of Eisenia foetida is slightly more than that of  the CF of  Perionyx excavatus.

Figure 4: Effect of time of incubation on procoagulant activity of CF of Eisenia foetida.   Click here to View figure

(Each value represents mean of three independent determinations)

Figure 5: Effect of time of incubation on procoagulant activity of CF of Perionyx excavatus. Click here to View figure

(Each value represents mean of three independent determinations)

The hemolytic and procoagulant activities may be due to different factors present in the CF of different species of earthworms as the CF of   Eisenia foetida exhibited procoagulant activity but not hemolytic activity whereas the CF of Perionyx excavatus exhibited both the activities. It also reveals the fact that different species of earthworms may comprise different molecules.

References

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