Abstract
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 x106 /g) in E.fetida and lowest (2.8± 0.7 x106 /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.
Highlights
1.0 Introduction Annelids are supposed to be the earliest animals in phylogenetic tree in which both cellular and humoral immune responses are developed
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
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 display active immune responses (Dhainaut and Scaps, 2011)
Summary
Collected earthworms were identified with the help of available literature (Stephenson, 1923; Gates, 1972; Julka,1988), later re-confirmed with amplified. Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, California) on an ABI3500 with LCO 1490 - HCO2198 primers (Xcelris Genomics Pvt. Ltd., Ahmedabad, India). 2.3 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. 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 [8,9,10] times. After collection of coelomic fluid in cold extrusion buffer, worms were released in soil. Adverting shortly to the presentation of data, all targeted earthworms species collected and identified from the study area are arranged family wise.
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