International Journal of Zoology and Applied Biosciences ISSN: 2455-9571 Volume 4, Issue 4, pp: 146-154, 2019 https://doi.org/10.5281/zenodo http://www.ijzab.com Research Article SURVEY AND PARTIAL CHARACTERIZATION OF AGGLUTININS IN THE HEPATOPANCREAS EXTRACT OF THREE MARINE GASTROPODS, TROCHUS RADIATUS (GMELIN, 1791), TURBINELLA PYRUM (LINNAEUS, 1767) AND BABYLONIA ZEYLANICA (BRUGUIERE, 1789) * Thana Lakshmi, K. Department of Zoology, Holy Cross College, Nagercoil - 629 004, Tamilnadu, India Article History: Received 22nd June 2019; Accepted 19th July 2019; Published 30th July 2019 ABSTRACT The hepatopancreas exctract of three marine gastropods, Trochus radiatus (Gmelin, 1791), Turbinella pyrum (Linnaeus, 1767) and Babylonia zeylanica (Bruguiere, 1789), were studied for the presence of agglutinins using 10 different mammalian erythrocytes. Babylonia zeylanica agglutinated all the 10 erythrocytes but in varying degrees. Agglutinins with strong affinity for rabbit and rat erythrocytes, as revealed by high haemagglutination (HA) tire, were observed in Trochus radiatus and Babylonia zeylanica. Hepatopancreas extract of Babylonia zeylanica also showed high affinity for pig erythrocytes (HA = 2048). Turbinella pyrum showed a weak activity against rabbit (HA = 8) and rat erythrocytes (HA = 2) but no haemagglutinting activity was observed against all the other erythrocytes. Results revealed the pH and temperature sensitivity of the, hepatopancreas agglutinins of all three species, as the HA titre varied with pH and temperature. Calcium ions at 10 mM concentration greatly enhanced the activity of the agglutinins of Trochus radiatus and Babylonia zeylanica but a slight decrease in activity was noticed for the agglutinin of Turbinella pyrum. The present work provides the basic information needed for further research on the isolation and purification of the agglutinins of Trochus radiatus and Babylonia zeylanica, The purified agglutinins can be studied for their pharmacological properties and tested for their biomedical potential in the diagnosis and treatment of several fatal and difficult-to-treat diseases. Keywords: Hepatopancreas exctract, Haemagglutination, Tris-buffered saline, Calcium ions. INTRODUCTION based resources have witnessed a paradigm shift in recent years. Scientists believe that an untapped reservoir of The rapid increase in population, changing human life style powerful new medicines is in the oceans. Although many and climate change impacts, have propelled the origin and of these products are not likely to become therapeutics, the spread of many incurable and fatal diseases like influenza, information gained from studying them is likely to lead to diabetes, coronary disorder, AIDS and cancer globally (Pati the development and understanding of novel molecular et al., 2015). Increasing emergence of multi-resistant targets, which in turn may lead to the development of new strains of pathogenic bacteria, as well as new epidemics, classes of therapeutic agents. i.e., dengue, chikungunya, bird flu, swine flu, drive the need to develop new therapeutics. As disease resistance to Marine organisms are a rich source of biologically antibiotics and other drugs continues to build, even new active compounds of interest for development of methods of discovery such as combinatorial chemistry may pharmaceuticals and alternative medicines (Benkendorff, not be able to meet the ever-increasing need for more 2009). Natural antibody like humoral substances efficient and more effective compounds. agglutinins, lysins and antimicrobial factors capable of agglutination, hemolysis and antibacterial properties have Oceans are considered as treasure house of valuable been reported in the body fluids of many invertebrates bioactive compounds. Exploration and exploitation of sea- (Moffett, 1995). Bioactive substances formed by marine *Corresponding Author: Dr. Thana Lakshmi, K, Department of Zoology, Holy Cross College, Nagercoil- 629 004, Tamilnadu, India. E mail: [email protected] 146 Thana Lakshmi, K. Int. J. Zool. Appl. Biosci., 4(4), 146-154, 2019 organisms such as protozoans, poriferans, cnidarians, marine life in the ocean, pharmaceutically valuable annelids, arthropods, molluscs and echinoderms have products derived from marine organisms are meager and attracted attention, due to their antiviral, antimicrobial, the marine resources remain under explored. It is hoped antiprotozoal, antifungal, antihelminthic and anticancer that the present work on agglutinins, would provide the activities (Zapata & Amemiya, 2000). basic information for new pharmaceutical discoveries from Molluscs, a wide group of invertebrates, constitute to marine gastropods, in order to fight against many fatal and about 23% of the animals inhabiting the marine difficult-to-treat diseases. hydrosphere among which 80% of them are gastropods. In fact a number of secondary metabolites from mollucs have MATERIALS AND METHODS been tested for their strong bioactive properties and have reported to be valuable pharmaceutical applications. Many Animals studied they have undergone preclinical assessment and some of The present study was carried out on three species of them have entered clinical trials. Dolastatin 10, Ziconotide marine gastropods collected Arockiapuram, located in the Neosurugatoxin, Diemenensin A, Chromodorolide A, southeast coast of Tamil Nadu. The species were identified Ulapualide A, Onchidal, KLH (Keyhole Limpet (ZSI, Chennai) as Trochus radiatus (Gmelin, 1791), Hemocyanin), Kahalalide F, Keenamide A, and Turbinella pyrum (Linnaeus, 1767) and Babylonia Bursatellanin P are some of the products derived from zeylanica (Bruguiere, 1789) were collected from marine gastropods. Considering the wealth in diversity of Arockiapuram, southeast coast of Tamil Nadu (Table 1). Table 1. Systematic position of the marine gastropods studied. Phylum Mollusca Class Gastropoda Subclass Vetigastropoda Caenogastropoda Caenogastropoda Order Trochida Neogastropoda Neogastropoda Superfamily Trochoidea Turbinelloidea Muricoidea Family Trochidae Turbinellidae Babyloniidae Trochus radiatus (Gmelin,1791) Turbinella pyrum (Linnaeus, Babylonia zeylanica 1767) (Bruguiere,1789) Species Preparation of Hepatopancreas extract Haemagglutination (HA) assay Animals collected were brought to the laboratory, rinsed Agglutinins are capable of agglutinating a variety of with sterile water to remove the debris and other adhering foreign particles such as bacteria, yeasts, protozoans, matter from outer surface of the shell and blotted to remove vertebrate erythrocytes. Erythrocytes are particularly, the water prior to the experiment. After the careful removal of useful targets as they are readily available and agglutination is observable even with the naked eye. Hence in the present shell, the whole body of each snail was removed and study, 10 different mammalian erythrocytes (human A, B washed thoroughly with normal saline solution and blotted. and O, rabbit, rat, dog, pig, cow, goat and buffalo) were The hepatopancreas was isolated from other tissues, used to studying activity of the agglutinins in the washed with sterile saline and an extract of hepatopancreas hepatopancreas extract of the marine gastropods. was then prepared by homogenizing 100 mg of the tissue in Haemagglutination assays were performed in 1 ml of sterile saline. Homogenized extract was centrifuged microtitre plates with ‘U’ bottomed wells, by serial two at 4000 x g for 10 minutes at 4°C and the supernatant was fold dilutions of 25 μl of the sample with an equal volume pooled in small aliquots and stored at -20°C for further of TRIS buffer (pH 7.5). After the dilution of the sample, assays. 25μl of 1.5% erythrocyte (from different mammals) 147 Thana Lakshmi, K. Int. J. Zool. Appl. Biosci., 4(4), 146-154, 2019 suspension was added, mixed well and incubated for 1h at molluscs have been tested for their strong bioactive room temperature. The HA titre was determined as the properties i.e., neuromuscular blocking action, anti- reciprocal of the highest dilution of the sample giving predator, antimicrobial, anti-neoplastic and cytotoxic complete agglutination. Since the agglutinins in the activity. The most promising metabolite isolated from a hepatopancreas extract of all the animals showed high marine mollusc is Dolastatin 10, an anti-neoplastic peptide affinity for rabbit erythrocytes, for further studies on isolated from the sea hare Dolabella auricularia (Pettit et characterization of the agglutinins, rabbit erythrocytes were al., 1987). Dolastatin 10 has recently reached clinical trials used in the hemagglutination assays. in the United States and is reported to be one of the most potent anticancer agents known (Carte, 1996). Ziconotide Haemagglutination assay for pH and thermal stability isolated from the the venom of the predatory Indo-Pacific To study the effect of pH on the haemagglutinating activity, marine molluscs, Conus magus shows remarkable analgesic 25 µl of the sample was suspended in 25 µl of Tris-buffered activity, 1,000 times more active that morphine in animal saline (TBS) of varying pH (6.5, 7.5 and 8.5) and serially models of nociceptic pain, is due to the blockage of diluted in a microtitre plate. It was incubated for 1 h at calcium channels (Olivera, 2000). Neosurugatoxin isolated room temperature and mixed with 25 µl of 1.5% rabbit from the Japanese ivory mollusc Babylonia japonica, is a erythrocyte suspension. The HA titre was determined as reversible antagonist of acetylcholine receptors (Ireland et before.