Comp. Biochem. Physiol. Vol. 97B, No. 2, pp. 321-325, 1990 0305-0491/90 $3.00+ 0.00 Printed in Great Britain Pergamon Pressplc CHARACTERIZATION OF DIVERSE HEMOLYMPH LECTINS IN THE COTTON CATERPILLAR SPODOPTERA LITTORALIS SOMAYA O. EL DEEB,* TAHANYH. HASSAN,*EDWIN L. COOPERt and ABDELHAKIMM. SAAD*~ *Department of Zoology, Faculty of Science, Cairo University, Cairo 12613, Egypt; and i'Department of Anatomy, School of Medicine, Center for the Health Sciences, University of California, Los Angeles, CA 90024, USA (Received 5 April 1990) Abstraet--l. Hemolymph lectins (agglutinins) of the cotton caterpillar Spodoptera littoralis were analyzed by agglutination, cross-absorption and carbohydrate-hemagglutination inhibition using several vertebrate erythrocytes. 2. Lectins were found to interact, with all tested erythrocytes, by binding to carbohydrate moieties but showing no definite specificity. 3. Disulphide bonds were probably absent as 2-ME treatment was ineffective. 4. By cross-absorption studies, we have proposed that the hemolymph contains multiple lectins. INTRODUCTION Body fluids of invertebrates contain naturally- occurring aggregations of bacteria (Arimoto and Trip, 1977; Pauley et al., 1971; Pistole, 1982) and erythrocytes (Cushing et al., 1963; Brown et al., 1968; Tyler, 1946; Yeaton, 1981). In view of their carbo- hydrate-binding capacity, insect agglutinins have been proposed to participate in the early recognition- ary steps in pathogen clearance from the hemocoel (Gupta, 1986; Renwrantz, 1983; Sharon, 1984). Agglutinins, by binding to surface glycoconjugates on foreign substances, may serve as a discriminatory link between non-self material and the hemocytes in- volved in phagocytosis or encapsulation reactions. Several models have been proposed for the role of agglutinins in insect defense responses (Bradely et al., 1989; Coombe et al., 1982; Ratner and Vinson, 1983; Renwarntz, 1983). These models hypothesize that hemocyte membrane-bound hemagglutinin serves to directly bind foreign particles, and humoral hemag- glutinins serve as opsonic factors that bind the foreign particles and facilitate their uptake by hemo- cytes. None of these models have been proven, and the role of hemagglutinin in insect defence mech- anisms is uncertain. The cotton caterpillar Spodoptera littoralis (Lepi- doptera, Noctuidae) is of vital economic importance since it attacks leaves, flowers, youngbolls and other tender parts of cotton plants. Despite this infor- mation on its living habits which are economically threatening, there is nothing known relative to the immune system of this insect. Therefore, we have characterized the hemolymph lectins of S. linoralis with respect to their (1) agglutination of different vertebrate erythrocytes, (2) erythrocyte absorption profiles and (3) carbohydrate specificities as deter- mined by inhibition of agglutination using defined :~Author to whom correspondence should be addressed. sugar and polysaccharides. Such information might reveal new approaches for control of this serious pest. MATERIALS AND METHODS Insects Successive generations of Spodoptera fittoralis (Lepi- doptera, Nictuidae) larvae, from a homogenous culture, were reared and maintained as described earlier (Flaschen- trager et aL, 1958). One-day post-final moult "zero-day" larvae were used throughout this study. Collection of hemolymph Hemolymph (HL) was collected by puncturing the larvae at the base of the second proleg. Extruded HL pooled from 20-25 larvae (1.5-2 ml) was centrifuged (3000 rpm, 10 rain, 4°C). The supernatant was removed and stored at -20°C until needed. Induction of hemagglutinins Larvae were either left intact (control) or injected (exper- imental) with 5/~1 of 5% vertebrate erythrocyte suspension in phosphate buffered saline (PBS, Ca2+ and Mg 2+ free, pH 7.2). Types of erythrocytes used were of human type A (HRBC), obtained in heparin (Sigma Chemical Company, St Louis, MO) from volunteers of the laboratory staff. Rabbit (RRBC), guinea pig (GRBC), rat (rRBC), and mouse (MRBC) blood samples were collected fresh on Alserver's solution. All blood samples were stored at 4°C and used within 48 hr. For assays, erythrocytes were cen- trifuged, 1500rpm for 10 rain, 4°C, the burly coat removed and washed three times in PBS, pH = 7.2. Hemagglutination assays Hemolymph agglutinating activity was titrated by mixing 2-fold serial dilutions of hemolymph (25/d) with equal volumes of PBS, pH = 7.2 in U-bottom microtiter trays. An equal volume (25/~1) of 1% erythrocyte suspension was added and agglutination evaluated after erythrocytes had settled at room temperature (3 hr). Titers were expressed as the greatest dilution (log2) of the sample causing complete agglutination. 321