Screening of Immunocompetent Coelomic Cells in Earthworms

This study describes digital diagnostics density of different coelomic cells in various species of earthworms. Immunocompetent coelomic cells, of different species of earthworms (Eisenia fetida, Eudrilus eugeniae, Eudichogaster prashadi and Perionyx sansibaricus) were isolated and subcultured with exposure of modified cold shock treatment. Study shows that concurrent density of amoebocyte and eleocyte of various species of earthworms can be maintained efficiently for in-vitro studies. The number of coelomocytes per gram of body weight was recorded highest (5.0± 0.8 x10 /g) in E.fetida and lowest (2.8± 0.7 x10 /g) in P.sansibaricus. A high percentage of autofluroscence was recorded in eleocyte of Eisenia fetida followed by Perionyx sansibaricus. Aggregates of pigmented granules and brown bodies were recorded almost in all granular amoebocytes and were significantly high in E. prashadi. Study may serve as useful aid in further immuno-cytochemical bioremediation studies and to decipher mechanism of uptake of by coelomic cells of earthworms.


Introduction
Annelids are supposed to be the earliest animals in phylogenetic tree in which both cellular and humoral immune responses are developed. Their body (coelomic) fluid consists various types of immunocompetent cells, the coelomocytes, which take part in various physiological processes including wound healing, blood coagulation, regeneration and other immune responses. These cells may expel out under stressful conditions through inter-segmental dorsal pores with increased intra-coelomic pressure. Various studies (Hosteller and Cooper, 1972;Engelmann et al., 2005) considered them as analogous to leukocyte as they are capable of phagocytosis and perform as function of macrophages. They also have natural killer cell feature, mediate lytic reactions against several targets and also secrete antimicrobial peptides (Porchet-Hennere et al., 1992;Cooper et al., 1995;Cooper, 2002;Cossarizza et al., 1996;Koros et al., 2002;Madhusudan et al., 2009). Usually earthworm Eisenia fetida was chosen as model organism for the immunological studies/ bio-remediating agent (Hamed et al., 2002; Somogyi, 2012) as they lack adaptive immunity (Fischer and Harvath, 1977) and also display active immune responses (Dhainaut and Scaps, 2011).
A uniform classification of coelomocyte of different species of earthworm is little difficult (Adomowicz and Wojtaszek, 2001) as they exists in various functional states and stages of maturation. Broadly on the basis of morphology, Cooper and Stein (1981) described two type of coelomocytes namely, amoebocytes (hyaline and granular) and eleocyte. Amoebocytes move by pseudopodia devour foreign material and are rich in lysosome. While elecocytes are rich in glycogen particles, lipid droplets and characterised by the presence of distinct yellow granules, chloragosomes. The origin and relationship of coelomocytes are not yet completely known. It was assumed that amoebocyte derived from mesenchymal lining of coelom, whereas, eleocytes originate by detachment of chloragogen cells covering intestinal tract (Affar et al., 1998;Hamed et al., 2002). Chlorogogenic cells are responsible for maintaining constant pH and ionic balance of both, coelomic fluid and haemolymph (Prento, 1979;Affar et al., 1998). Cooper and Stein (1981) suggested similar origin of hyaline and granular amoebocyte. However, no unambiguous relation was recorded between them. Amoebocyte participate in the transport and storage of nutritive substances (Valembois and Cazaux, 1970), wound healing (Byzowa, 1974), cellular defence reactions (Cooper, 1996), phagocytosis (Stein and Cooper, 1981;Bilej et al., 1990;Dales and Kalac, 1992;Ranzelli-Cain and Kaloustein, 1995;Cossarizza et al., 1996), encapsulation and modulation (Valembois et al., 1994). While, eleocytes play an important role in immune responses producing bactericidal substances (Valembois et al., 1982;Ville et al., 1995;Milochau et al., 1997) and also participates in reaction of encapsulation and formation of brown bodies (Cooper and Stein, 1981). The number and composition of the coelomocyte depends on exogenous (environmental) as well as endogenous (biotic, life cycle) factors. The interest to study coelomocytes of earthworms as immunocompetent cell is increasing worldwide. Not much work has been carried out to categorize coelomocyte in different species of earthworms. Therefore, the present study was undertaken to perform rapid screening of different coelomocytes, their isolation and sub-culturing of different species of earthworms to investigate their immunocytochemical efficiency in soil system.

Collection of earthworms
Four species of earthworms viz., Eudichogaster prashadi (family Octochaetidae), Perionyx sansibaricus (family Megascolecidae), Eisenia fetida (family Lumbricidae) and Eudrilus eugeniae (family Eudrilidae) were collected by digging and hand sorting method from botanical garden, agriculture land and reserved forest area of Sagar MP, India during August -September, 2014. For identification, collected specimens were preserved in ethyl alcohol for molecular characterization, and also fixed in 4% formalin for morpho-anatomical study. Coelomic cells were extracted from live earthworms and subcultured in CO2 incubator.

Isolation of Coelomocytes
Collected worms were thoroughly washed in running tap water before rinsing in distilled water and were not subjected to any control condition. Worms were placed on wet cotton to ensure complete defecation in order to avoid contamination during harvesting of coelomocytes. After 2-3 hrs, worms were wiped with cotton wool soaked with 70 % ethyl alcohol to avoid any further contamination. The surface cleaned worms were placed alternately in sterile petridish containing cold extrusion buffer (NaCl 71.2mM; Ethanol 5%; Guaicol-glycerol-ether 50.4mM; EGTA 5mM, pH 7.3) and distilled water at interval of one minute for 8-10 times. Coelomic fluid extruded out through dorsal pores due to external stress condition. After collection of coelomic fluid in cold extrusion buffer, worms were released in soil.

Culturing of Coelomocytes
The excreted coelomic fluid was pipette into tubes filled with LBSS solution (NaCl 71.5mM; KCl 4.8mM; MgSo4.7H2O 1.1mM; KH2PO4 0.4mM, pH 7.3) and centrifuged at 4°C for 5 min. Loose pellets of coelomocytes were washed 2-3 times with cold LBSS solution. Cell count was maintained 10 7 /ml with trypan blue exclusion. The isolated coelomocytes were loaded in petridish with DMEM supplemented with 10% FBS and incubated for 3 days in CO2 incubator.

Cell Viability
Viability of cells was recorded at the time of isolation and after incubation for three days using haemocytometer and was examined in phase contrast/fluorescence microscope.

Statistical Analysis of Data
Three replicates were considered for statistical analysis. Student's t-test was applied to observe level of significance of cell viability.

Results and Discussion 3.1 Details of earthwormsspp.
Adverting shortly to the presentation of data, all targeted earthworms species collected and identified from the study area are arranged family wise. Each entry gives the information in sequence; scientific name of earthworm species, locality with collection number in parenthesis, date of collection, examined morpho-anatomical characters and sample no. Clitellum on segments xiv-xviii. Male pores minute, paired at the tip of penes, retractable into copulatory chambers, apertures of copulatory chambers large transverse slits on segment 17, just in front of inter-segmental furrow 17/18. Female pores paired, large transverse slits, close to sides of the body, on segment xiv. Y-shaped gland present which opens though own porophore into copulatory chamber. Sample no examined for molecular characterization: EW71-A-25 (Fig 9).

Coelomocytes in different species of earthworms
Different coelomocytes of Eisenia fetida; Eudichogaster prashadi; Perionyx sansibaricus and Eudrilus eugeniae were observed and illustrated in Fig 5 to 8. A high percentage of autofluroscence was recorded in eleocyte of E. fetida (Fig 5d) followed by P. sansibaricus (Fig 7d). Aggregates of pigmented granules, brown bodies were recorded almost in all granular amoebocyte and significantly high in E. prashadi (Fig 6a). Precise reason of their increase in particular species is unknown, but it may indicate their efficiency of phagocytosis and encapsulation activity of invading bacteria/particulate waste. A remarkable divergence in fluorescence granules in eleocyte was detected in different earthworm species and a very high density was recorded in E. fetida. andrei and these brown aggregates were also recorded significantly high in E. Prashadi (Fig 6a) in present study. This may be resulted from the insolubilization of oxidised organic substrates (last stage of catabolism and segregation of unwanted material in granular amoebocytes as suggested by Cholewa et al., 2006). It can be speculated that free floating amoebocytes may shed from the degraded eleocytes. The observed fluorescence material may be due to flavins (Aubin, 1979 in coelomic fluid containing free wandering coelomocytes. It was observed that hyaline amoebocytes in all species of worms have numerous pseudopodia and are distributed in the cell periphery with short lobopodia (Fig 5b, 6b, 7b, 8b). Adomowicz and Wajtaszek (2001) differentiated these amoebocyte into AI (regularly distributed numerous pseudopodia on the cell periphery and with short lobopodia); and AII (irregularly distributed pseudopodia, often at one pole of cell with long filopodia). These differentiations were not observed in present study. All authors agreed to call them inclusively amoebocyte rather than I and II. As the formation of pseudopodia, the shape of the nucleus and the granular cytoplasm represents the maturity and metabolic activity of amoebocyte.
A gradual increase in cell density from the time of isolation to subculturing was recorded in all species. After incubation of three days the highest total cell count (8.2± 0.6 x10 6 /g) was observed in E.fetida followed by E.prashadi (6.2± 0.6 x10 6 /g). Number of autofluroscent eleocytes increased significantly in subculturing, while the subculturing of hyaline amoebocyte were not obtained in optimum count. The culture plate of proliferating cells in present study didn't exhibit any bacterial or fungal contamination, even though, any antibiotic /antifungal agent was not used in the present study. Some protozoan contamination at the stage of isolation was recorded but was subsided with future culturing and subculturing. The modified protocol used in present study revealed that the coelomocytes can be successfully isolated and cultured with the application of cold shock treatment. Also, subculture can be used for further immunocytochemical studies. The percentage of various coelomocytes may be species specific, but seasonal environmental stress may not be ignored. At present, we cannot exclude possibility of variation in number and size of coelomocytes in relation to availability of nutritive compounds as well as their annual cycles. The study can serve as useful aid to decipher the mechanism of uptake and scavenging of pollutants by coelomocytes of different species of earthworms' in-vitro.

Acknowledgement
We acknowledge the financial support of the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, New Delhi, to carry out this study.

Competing Interest
The authors declare that they have no competing interests.   The evolutionary history was inferred using the Neighbor-Joining method [1]. GenBank accession description of the sequences from earlier studies was shown in the phylogenetic tree along with bootstrap test (1000 replicates) next to the branches [2]. Sepia aculeata COI gene, cuttlefishes (Mollusca) was used as out group. Bar 5 changes/100 characters. Evolutionary analyses were conducted in MEGA v6 [3].