Carbohydrate-binding plasma proteins from the gastropod Biomphalaria glabrata: Strain specificity and the effects of trematode infection

Developmental and Comparative Immunology, Vol. 16, pp. 355-366, 1992 0145-305X/92 $5.00 + 00 Printed in the USA. All rights reserved. Copyright © 1992 Pergamon Press Ltd.

CARBOHYDRATE-BINDING PLASMA PROTEINS FROM THE GASTROPOD Biomphalaria glabrata: STRAIN SPECIFICITY AND

THE EFFECTS OF TREMATODE INFECTION

Fernando Monroy, Lynn A. Hertel, and Eric S. Loker

Department of Biology. University of New Mexico, Albuquerque, NM 87131 (Submitted June 1991; Accepted November 1991)

[]Abstract--As lectins are believed to mediate non-self-recognition in molluscs, carbohydrate- binding proteins (CBP) from the circulating plasma of the gastropod Bioraphalaria glabrata were harvested by affinity chromatography using six different monosaccharides as ligands. Pools of plasma were derived from B. glabrata of either the M line strain, which is susceptible to infection with the PR1 strain of the digenetic trematode Schistosoma mansoni, or from the 13-16-R1 strain, which is resistant to infection. For each strain, plasma was obtained from control snails and from snails exposed to infection 1 or 8 days previously with S. raansoni or the related digenean, Echinostoma paraensei, which is able to develop in either host strain. For control snails, only minor interstrain differences were noted. In M line snails exposed 8 days previously to either parasite, marked changes in CBP populations were observed. Only E. paraensei infections produced comparable alterations in 13-16-R1 snails. The most conspicuous changes noted were the increased production of 80-120 kDa CBP in both strains, 150-210 kDa in 13-16-R1 snails, and 190-210 kDa in M line snails. The results demonstrate 1) interstrain differences in CBP, particularly following exposure to trematodes; 2) that infection provokes increased production and diversity of CBP that bind with greater affinity to the columns; and 3) that snails of the same strain respond differently to the two parasites used.

[Keywords--Molluscs; Biomphalaria glabrata; Digenetic trematodes; Schistosoma mansoni; Echinostoma paraensei; Comparative immunobiology; Hemolymph proteins; Lectins.

Address correspondence to Dr. Eric S. Loker.

Introduction

Non-self-recognition in invertebrates is accomplished without the benefit of im- munog lobu l in mo lecu l e s (1). Lec t i n s have been repeatedly implicated as prob- able functional analogs of immunoglobulins in invertebrates , including molluscs (2-8). Lect ins or lectin-like molecules are known to be present in several gas- t ropod species (4,7), and can be recov- e red f rom the h e m o l y m p h , a l b u m e n gland, egg masses , and body mucus . Several lectins have been isolated f rom the f reshwater snail Biomphalaria glabrata (9-13).

A lectin in the p lasma of B. glabrata has been found to facilitate phagocytosis of yeas t by h e m o c y t e s (14). Fur ther- more, this opsonin was present in two strains (the 10-R2 and 13-16-R1 strains) of B. glabrata resistant to the digenetic t r e m a t o d e Sch i s tosoma manson i and was lacking in the s c h i s t o s o m e - s u s - ceptible M line strain. The opsonin f rom resistant snails has yet to be character- ized, although a 57 kDa plasma protein has been implicated as a possible candi- date (15). A 55 kDa polypeptide f rom the p lasma of res is tant snails binds to S. mansoni sporocysts (16); a comparable molecule was lacking in p lasma f rom susceptible snails. A hemagglutinin, also 55 kDa, f rom the albumen gland and he- m o l y m p h of s c h i s t o s o m e - s u s c e p t i b l e B. glabrata was found to bind to S. mansoni miracidia, sporocysts , and cercariae (10); the authors postulated that it may

355

356 F Monroy, L. A. Hertel, and E. S. Loker

mask larval trematodes from the cellular defense system of their host.

Prior attempts to document changes in plasma proteins following infection of both susceptible and resistant snails of B. glabrata with S. mansoni (13,17) have, in general, shown only subtle changes. In contrast, infection of B. glabrata with another digenean, Echino- stoma paraensei, resulted in conspicuous qualitative and quantitative alter- at ions of hemolymph polypept ides . Hemolymph of snails infected with E. paraensei contained increased quantities of polypeptides of 80-120 kDa and 200 kDa (18). The 80-120 kDa polypeptide (or family of polypeptides) can be puri- fied by affinity chromatography using L-fucose-conjugated agarose beads and has carbohydrate-inhibitable agglutinat- ing activity for rabbit erythrocytes (13), suggesting it has the properties of a lectin. This molecule resembles closely an 89-105 kDa galactose-inhibitable hemagglutinin p rev ious ly isolated from plasma of M line B. glabrata by Boswell and Bayne (12).

Much additional study is required to characterize and elucidate the functional roles of snail plasma molecules with the properties of lectins. Here we further characterize carbohydrate-binding proteins (CBP) in the plasma of B. glabrata using affinity chromatography. Six similar affinity columns, each containing a mixture of beads conjugated to different monosaccharides, hereafter referred to as sugars, were used to collect CBE Six different sugars were used as primary column eluants. The compositions of CBP in each of two snail strains and alterations that occurred following exposure to S. mansoni or E. paraensei are documented.

Materials and Methods

Animals

The M line and 13-16-R1 strains of B. glabrata and the digenetic trematode E.

paraensei were maintained as described by Loker and Hertel (18), and S. mansoni was maintained as described by Stibbs et al. (19). M line snails are susceptible to S. mansoni whereas 13-16-RI snails are resistant (20); E. paraensei can successfully infect both snail strains, although M line snails are more susceptible to this parasite than 13-16-RI snails (unpublished observations). E. paraensei was maintained in hamsters and M line snails, and the Puerto Rican 1 strain of S. mansoni was maintained in outbred mice and M line snails. E. paraensei miracidia were hatched from eggs dissected from adult worms that were taken from the small intestine of hamsters; miracidia of S. mansoni were hatched from eggs derived from homogenized mouse livers.

Snail Exposures

Juvenile (6 mm shell diameter) M line and 13-16-R1 snails were selected from stock tanks and exposed individually to 10-15 freshly collected miracidia of E. paraensei or S. mansoni for 3 h, in wells containing 2 mL of water. Control snails were subjected to the same treatment but without miracidia.

Affinity Chromatography Columns

Each of six disposable Poly-Prep c h r o m a t o g r a p h y co lumns (BioRad, Richmond, CA) was packed with a total of 1.2 mL of a mixture of monosaccharide-conjugated agarose beads. The bead mixture contained equivalent volumes of beads conjugated to L( -- )fucose, alpha- methyl D( +)mannoside, D( + )mannose, D( + )glucose, N-acetyl-D( + )glucos- amine (GluNAc), or N-acetyl-o(+)ga- lactosamine (GalNAc). All conjugated beads were from the Sigma Chemical Company, St. Louis, MO. Columns were washed extensively in calcium buffer (CB, 0.05 M Tris-HC1, pH 7.2, contain-

B. glabrata plasma proteins 357

ing 0.02 M CaCI2) and stored at 4°C in CB with 0.02% sodium azide until used.

Experimental Design

For both strains, unexposed control snails and snails exposed to S. rnansoni or E. paraensei 1 or 8 days previously were examined. Hemolymph from approximately 50 snails/group was pooled and held at 4°C during collection. Pooled hemolymph was centrifuged (110 x g for 10 min) to remove hemocytes and debris and the resultant plasma (approximately 700-800 txL) was mixed with an equal volume of CB and divided into six equal aliquots. Each of the six affinity columns, which were held at 4°C, received one such aliquot. The plasma sample was allowed to seep into each column and was held among the column beads for 15 min, then the column was washed with approximately 10 mL of CB to remove extraneous proteins.

The following elution strategy was used with the six columns. For the first column, 1 mL of a 1.0 mM solution of L-fucose, designated as the primary sugar for this particular column, was added. This and other sugar-containing solutions specified below were made up in CB. The column eluate from this ap- plication, referred to as the 1.0 mM eluate, was collected directly in Centricon ultrafiltration units (Amicon, Beverly, MA) with I0 kDa cutoff. The column was then rinsed with 10 mL of CB. Then, 1 mL of a 100 mM solution of L-fucose was applied to the column and the eluate, designated as the 100 mM eluate, was collected as described above. The column was then washed with at least 10 mL of CB. One mL of a mixture of five carbohydrates (D-galactose, D-mannose, D-glucose, GIuNAc, and GalNAc, each at I00 mM) was added and the eluate collected similarly. This was designated the mixed sugar eluate. After washing the column again with CB, I mL of 2 mM EDTA in 50 mM Tris-HCl, pH 7.2, was

added to the column, and this eluate, called the EDTA eluate, was collected. Finally, the column was washed extensively with CB and stored as described above.

Procedures for the remaining five columns were similar except that each column used a different primary sugar (D- galactose, D-mannose, D-glucose, GIu- NAC, or GalNAc). For each column, the mixture of five sugars added to the column lacked the primary sugar for that column. Thus, for each pool of plasma, a total of 24 column fractions was gener- ated, four for each of the six columns used.

Column fractions were concentrated approximately twentyfold and, as con- cent ra ted sample volumes differed slightly following ultrafiltration, the volumes were adjusted such that all were equivalent. Samples were then subjected to SDS-PAGE on 9% gels using the Bio- Rad mini-Protean II electrophoresis cell. General SDS-PAGE protocols and staining procedures were as previously described (18). For each of the groups of snails specified above, at least two pools of plasma were subjected to this protocol.

Results

Carbohydrate Binding Proteins in Plasma From Control Snails

The series of six affinity chromatography columns containing mixed carbohydrate beads was used to harvest CBP from pooled plasma derived from control snails of both M line and 13-16-R1 strains of B. glabrata. A typical result obtained for 13-16-R1 control snails is shown in Fig. 1A; results for only one of the six columns are shown here as the other columns yielded the same basic pattern of bands. Wash material obtained from the column just before addition of the primary sugar contained trace amounts of a 92 kDa polypeptide and hemoglobin

358 F. Monroy, L. A. Hertel, and E. S. Loker

200- -

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! 2 3 4 1 2 3 4 Figure 1. (A) SDS-PAGE profiles of eluates obtained from an affinity column loaded with pooled plasma obtained from control 13-16-R1 snails. Results obtained for the six columns loaded with the same pool of plasma were similar. The lane designations are: 1) the 1 mM primary sugar eluate; 2) the 100 mM primary sugar eluate; 3) the mixed sugar eluate; and 4) the EDTA eluate. Positions of molecular weight standards, in kDa, are indicated. Faint bands appearing at 60-64 kDa (especially in lane 2) on this and other gels are extraneous and not derived from B. glabrata plasma. (B) This is similar to (A) except that pooled plasma from control M line snails was originally loaded onto the column.

(data not shown). A considerable diversity of bands was eluted by a 1.0 mM solution of the primary sugar. Prominent among these were bands of >200, hemoglobin (190 kDa), a diffuse band centered at 92 kDa, and a cluster of minor bands centered at 50 kDa. Hemoglobin, which is the most abundant protein in B. glabrata plasma, consistently bound to the columns but was much less abundant than in equivalent volumes of plasma. Minor bands could also be seen between 40 kDa and the dye front. Subsequent treatment with 100 mM solutions of the primary sugar generally eluted very little except small amounts of hemoglobin from the column. The mixed sugar eluate typically contained two faint, diffuse bands of approximately 92 kDa and 110- 120 kDa, and a faint band of 55 kDa. EDTA eluates contained a prominent band of 40 kDa, and occasionally some additional minor bands. Other differences in elution profiles obtained using different primary sugars are discussed below.

The patterns obtained with control M line snails resembled those for 13-16-R1, but some differences were noted (Fig.

1B). Once again, a prominent diffuse band at 92 kDa was eluted by a 1.0 mM solution of each primary sugar, and 100 mM of each primary sugar typically removed very little from the columns (although the example illustrated shows a faint band at 92 kDa). Mixed sugars generally removed a band of 92 kDa and an indistinct band at 110-120 kDa, and EDTA removed a prominent band of 40 kDa. The 55 kDa band observed in 13- 16-R1 preparations was lacking in M line eluates.

Effects o f Exposure to Trematodes on Plasma CBP

The effect of exposure to trematode larvae on the CBP in B. glabrata plasma was evaluated. Figure 2 illustrates repre- sentative results obtained for 13-16-R1 snails. Patterns resulting from use of different primary sugars are presented in the section below. Plasma taken from 13- 16-R1 snails at 1 day postexposure (dpe) to either trematode species did not differ noticeably from control plasma with respect to CBP elut ion profi les (Fig.


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I 2 3 4 1 2 3 4 Figure 2. SDS-PAGE profiles of eluates obtained from columns loaded with pooled plasma derived from 13-16-R1 snails: (A) 1 dpe to S. mansoni; (B) 1 dpe to E. paraensei; (C) 8 dpe to S. mansoni; and (D) 8 dpe to E. paraensei. Lane designations are as indicated in Fig. 1. Positions of molecular weight standards are indicated.

2A,B). As with control snails, most polypeptides were present in the 1.0 mM primary sugar eluate. In some cases, small amounts of the 92 kDa band were also found in the 100 mM eluate, but this was not typical.

Exposure to S. mansoni for 8 days did not have a pronounced effect in 13-16-R1 snails. Indeed, the elution profiles observed at 8 days were essentially indis- tinguishable from controls (Fig. 2C).

At 8 dpe to E. paraensei, conspicuous differences in elution profiles between

control and exposed 13-16-R1 snails were observed (Fig. 2D). A broad band of 150-210 kDa was noted in several of the 1.0 mM primary sugar eluants. This band was also prominent in 100 mM eluants, and depending on the primary sugar used (see below), also appeared in the mixed sugar and EDTA eluants. This band migrated similarly to hemoglobin, but could readily be distinguished by its black color following silver staining; hemoglobin typically stains red or brown and migrates as a focused band. Addi-

360 F. Monroy. L. A. Hertel, and E. S. Loker

tionally, increased amounts of material in the 80-120 kDa region were eluted by a 100 mM solution of the pr imary sugar: this molecular weight range was also represented in mixed sugar eluants and was much more abundant than in the comparable eluants f rom control snails. Mole- cules of this size were also frequently represented in the EDTA eluates.

Plasma taken f rom M line snails 1 dpe to ei ther t rematode species also did not differ conspicuously from control p lasma (Fig. 3A,B). In some cases, small quantities of the 92 kDa CBP were present in 100 m M pr imary sugar eluants, but oth-

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erwise there was little difference from controls.

E x p o s u r e to both t r e m a t o d e s provoked noticeable differences 8 days later in CBP in M line snails (Fig. 3C,D). The abundance of the 92 kDa band was en- hanced in 1.0 mM primary sugar eluants for both S. mansoni- and E. paraensei- exposed snails, but was especially prominent in the latter. The same band was also more likely to appear in 100 mM eluants as well. A broad, indistinct black band between 48-63 kDa was also some- times noted in 100 mM eluants. As with controls, bands of 92 kDa, and l l 0 -120

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Figure 3. SDS-PAGE profi les of eluates obtained from columns loaded with pooled plasma derived from M line snails: (A) 1 dpe to S. mansoni; (B) 1 dpe to E. paraensei; (C) 8 dpe to S. mansoni; and (D) 8 dpe to E. paraensei. Lane designations are as indicated in Fig. 1. Positions of molecular weight markers are indicated.


kDa appeared in the mixed sugar eluants, but they were more abundant than in control samples.

A 66 kDa band eluted with EDTA from columns loaded with plasma from S. mansoni-exposed snails. For E. paraensei-exposed snails, a CBP slightly heavier than hemoglobin, with a molecular weight of approximately 190-210 kDa, was also clearly present in both 1.0 and 100 mM primary sugar eluants. This band could also be distinguished from hemoglobin by its black color. The elution patterns of many of these bands were complex and are discussed further below. Although the figures provided imply that the amount of CBP derived from 13-16-R1 snails at 8 dpe to E. paraensei was greater than for M line snails, this was not a consistent pattern.

Elution of Prominent Bands by Different Primary Sugars

Many of the CBP eluted from the columns when a 1.0 mM solution of the primary sugar was added, regardless of the primary sugar used. For example, bands of >200, hemoglobin, and the cluster of bands at 55 kDa always mostly eluted with a 1.0 mM solution of any of the primary sugars. A band of 40 kDa typically was present in small quantities in all eluates, but was usually most abundant in the EDTA eluates, regardless of the primary sugar used, of the snail strain, or of infection status of the snails used.

The most interesting molecules with respect to primary sugar-specific elution patterns were three groups of broad, in- distinctly bounded bands, all of which stained black or dark gray. The first, designated as the high molecular weight group, consisted of the 150-210 kDa band of 13-16-R1 snails and the 190-210 kDa band of M line snails. Members of this group were only prominent in snails with 8-day E. paraensei infections, although minor quantities could be ob-

served in control snails. For 13-16-R1 snails, the 150-210 kDa band was mostly eluted by 1.0 mM and 100 mM solutions of each of the six primary sugars except GluNAc or GalNAc (Fig. 4A). A similar pattern was obtained for M line snails (Fig. 4B). GalNAc was almost totally in- effective in removing this high molecular weight material from the columns.

The second group is referred to as the 80-120 kDa complex. For snails of both strains, for both control and trematode- exposed snails, the most prominent com- ponent of this complex is the 92 kDa band. For control snails, this band was almost completely eluted by a 1.0 mM solution of any of the primary sugars tested (Fig. I). In E. paraensei-infected 13-16-R1 snails, however (Fig. 4A), the 92 kDa band was also prominently eluted by 100 mM solutions of all the primary sugars used, with the exception of Glu- NAc. GluNAc was relatively inefficient in eluting this band as judged by its relative abundance in the mixed sugar eluate. In M line snails, the 92 kDa band, in both control and trematode-exposed snails, is always best eluted by 1 mM solutions of the primary sugar (Fig. 4B).

The 110-120 kDa band, included as a member of the 80-120 kDa complex, was usually most abundant in the mixed sugar eluants, but it was successfully eluted by 100 mM GalNAc or especially by 100 mM GluNAc (e.g., Fig. 4A). The l l0-120 kDa band showed some ten- dency to resolve as two separate bands (Fig. 4B and C), suggestive of further heterogeneity within this complex. Fi- nally, a faint, indistinct band centered at approximately 85 kDa, separate from the prominent 92 kDa band, often appeared in EDTA eluates (e.g., Fig. 4B) and was considered a part of the 80-120 kDa complex.

The third group of molecules, visible only in eluates from M line snails with 8-day S. mansoni or especial ly E. paraensei infections, migrated as a very indistinct band between 48 and 63 kDa.

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Discussion

The approach described here repre- sents an attempt to harvest CBP from the hemolymph ofB. glabrata. The rationale for pursuing this goal is the belief that circulating polypeptides with the properties of lectins may be an important com- ponent of the internal defense system of this and other invertebrates (1-8). Col- umns containing beads conjugated to several different sugars were used so that as many different CBP as possible would bind to the columns. Also, by using different primary sugars to elute CBP, some inferences could be made regarding any sugar-specific behavior of the different CBP obtained. Use of both low and high concentrations of each primary sugar allowed a preliminary assessment of the affinity by which particular CBP bound to the columns. Later elution of each column with mixed sugars and EDTA allowed further characterization of particular CBP bound to each column. Some CBP, including some 92 kDa material, did not bind to the columns, or may not have been removed from the columns by the sequence of elution buff- ers used, and the results should be inter- preted with the view that the CBP collected by the specified protocols may not represent all CBP in the plasma ofB. glabrata. Results obtained for plasma from different batches of snails are comparable, however, as the same protocol was followed for all, and the order in which samples from different biological sources was applied to the columns was random. Furthermore, when different column runs were performed using plasma from snails of the same strain and/or exposure

regimen, the results were similar even if the replicate runs were separated in time by several weeks.

Using the specified protocols, several different polypeptides bound to, and were subsequently eluted from, the columns. The CBP recovered represented only a subset of the complex array of polypeptides present in B. glabrata plasma [for example, see Figs. 1-4 of (18)]. Particularly for control snails, the majority of CBP were eluted by 1 mM solutions of any of the primary sugars used, implying they do not bind with great affinity to any of the sugars present on the beads. The most prominent molecules among these was a broad band centered at 92 kDa. The 92 kDa band is very reminiscent of the B. glabrata 89- 115 kDa agglutinin described by Boswell and Bayne (12) and the 80-120 kDa agglutinin of Couch et al. (13).

Generally, 100 mM primary sugar solutions removed very little, if anything, from columns loaded with plasma from control snails, implying the absence of CBP with high binding affinity for the ligands represented on the beads. Mixed sugars eluted more material from such columns. One possible explanation is that the total number of sugar molecules in such eluates is five times higher than for the 100 mM primary sugar eluate, thereby providing a greater impetus for elution of CBE Alternatively, some of the CBP may be most efficiently eluted by multiple sugars. Other bands, most notably one of 40 kDa, were not fully eluted by any sugar and could be removed only by EDTA, suggesting a strong dependence on divalent cations for binding to the columns.

For plasma from control snails, little interstrain difference was noted. How- ever, a faint 55 kDa band was noted in mixed sugar eluates from S. mansoni- resistant 13-16-R1 snails that was not ob- viously present in S. mansoni-susceptible M line snails. This molecule

364 F. Monroy, L. A. Hertet, and E. S. Loker

may correspond to the 55 kDa polypeptide from B. glabrata reported by Spray and Granath (16) and Stein and Basch (10) to bind to S. mansoni sporocysts. Fryer et al. (15) tentatively implicated a molecule of similar molecular weight as a possible plasma opsonin from schistosome-resistant snails. In the present study, this molecule showed no obvious alteration in abundance following exposure to either trematode species, but its relatively scarcity may have prevented detection of such changes.

Bretting et al. (11) isolated a lectin from egg masses of B. glabrata with a molecular weight of 67 kDa; it dissoci- ated into subunits of 15-17 kDa on SDS- PAGE. In the present study, CBP of 15- 17 kDa were not observed in SDS-PAGE gel lanes. However, our study used B. glabrata plasma rather than egg masses as the starting material.

At 1 dpe to either trematode, CBP from snails of either strain showed no consistent alterations. One day may be too short an interval for the snails to respond detectably to infection; similarly, Couch et ai. (13) noted no obvious changes in agglutination titers in B. glabrata plasma at 1 dpe to either S. mansoni or E. paraensei. By 8 dpe, conspicuous differences in CBP elution patterns were noted; M line snails responded noticeably to both parasites whereas 13-16- R1 snails responded only to E. paraensei infections. The failure of S. mansoni to provoke noticeable alterations in 13-16- R1 snails may be attributable to the well- known inability of this parasite to develop in 13-16-R1 snails (20).

Both strains, but most noticeably 13- 16-R1 snails, responded to infection with E. paraensei by producing increased amounts of material in the 80-120 kDa complex, often in all four eluates from a particular column. Similar consequences of E. paraensei infection have been noted previously (13,18). The 80-120 kDa complex is difficult to characterize

precisely, owing to its diffuse nature, but in both strains it can be subdivided into at least three bands. These subregions can also be detected in plasma from control snails, but are much more obvious in plasma from infected snails. The most prominent of these is centered at 92 kDa and, in M line snails, is mostly eluted by 1 mM solutions of the primary sugar. In 13-16-R1 snails, it is eluted by both 1 mM and 100 mM solutions of the primary sugar as well as by mixed sugars.

Monosaccharide-specific elution was evident for both the 92 and 110- 120 kDa bands, especially for 13-16-R 1 snails. For example, GluNAc was relatively inefficient in eluting the 92 kDa band but was noticeably more successful in eluting the 110-120 kDa band.

One of the puzzling aspects of this study was that CBP were frequently eluted from the columns in a nonspecific manner. Certainly one explanation, al- luded to above for plasma from control snails, is that some of the polypeptides collected bind sugars, but only weakly, such that they are eluted by any sugar. However, for CBP from infected snails, a l ternat ive explanat ions have to be sought. For example , regarding the prominent 92 kDa band from 13-16-R1 snails, its elution behavior becomes much more complex in plasma samples from infected snails. One explanation for this change is that because it is more abundant, the standard elution volumes are insufficient to remove it completely from the columns. However, use of increased volumes of ! mM solutions of the primary sugar did not remove all of the 92 kDa material (personal observations). Subsequent treatment of the columns with 100 mM primary sugar solutions removed additional 92 kDa material, a pattern not seen in control snail plasma, suggesting that infection induced a qualitative shift towards production of material with greater binding affinity. Relative to control plasma sam-


pies, often considerably more of the 92 kDa material in plasma from infected snails was eluted only by mixed sugars, implying a more heterogeneous compo- sition of material of 92 kDa from such snails. This heterogeneity may effec- tively serve to mask monosaccharide- specific behavior.

A noticeable response to E. paraensei infection was also noted for 150-210 kDa molecules in 13-16-R1 snails, and in 190- 210 kDa molecules in M lines. S. mansoni provoked no comparable response, even in M line snails. For both strains, GIuNAc eluted relatively little, and GalNAc essentially none, of this material. It seems likely that this high molecular weight material is in some way related to the 80-120 kDa complex. Both groups of molecules produce diffuse bands, are gray-black on silver-stained gels (in contrast to most B. glabrata plasma components which stain brown- red), and react with polyclonal antisera raised in rabbits to the 80-120 kDa band (unpublished observations). The high molecular weight material may be modi- fied to yield molecules of 80-120 kDa. Similar considerations may hold for the indistinct material of 48-63 kDa present in plasma of infected snails.

In conclusion, the results imply that B. glabrata plasma contains a diversity of CBP that do not show conspicuous sugar-specific elution profiles. Inter- strain differences were noted in CBP profiles of control and especially trematode-infected snails. The two trematodes employed provoked different responses from snails of the same strain. Some ev- idence was obtained to suggest that in-

creases in affinity of CBP binding and in CBP diversity may follow exposure to trematode larvae.

The functional role(s) of the CBP detected here await further study. Of particular interest in this context are the faint 55 kDa 13-16-Rl-specific molecule also noted by other authors (10,15,16) and the three groups of diffuse banding, gray-black staining molecules specified above. Also of interest is the location of synthesis of CBP that increase in abundance in infected snails. It seems un- likely that these CBP are derived from developing parasites, as molecules with similar properties can be identified in the plasma of uninfected snails and because excretory/secretory products derived from cultured E. paraensei sporocysts and rediae do not contain similar molecules. More likely sources of trematode- induced CBP are circulating hemocytes, which increase sharply in abundance following exposure to E. paraensei (18). Also, B. glabrata hemocytes have been shown to secrete a diversity of different polypeptides (21), some of which resem- ble components of the 80-120 kDa complex discussed in the present paper [see Fig. 1 of (21)]. Finally, further study is required to understand the underlying factors responsible for heterogeneity of CBP, especially those of the 80-120 kDa complex.

Acknowledgements--We thank Ms. Lee Couch for expert technical assistance, and Dr. Christopher J. Bayne and Dr. Sarah E. Fryer for critically reading the manuscript. This study was supported by NIH grant AI- 24340.

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Carbohydrate-binding plasma proteins from the gastropod Biomphalaria glabrata: Strain specificity and the effects of trematode infection