NIH Public Access Author Manuscript Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1.

NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Dev Comp Immunol. 2010 April ; 34(4): 425. doi:10.1016/j.dci.2009.12.001.

Identification of components of egg masses indicates parental investment in immunoprotection of offspring by Biomphalaria glabrata (Gastropoda, Mollusca)

Jennifer J M Hathaway1, Coen M. Adema1,*, Barbara A. Stout1, Charlotte D Mobarak2, and Eric S Loker1 1 Center of Evolutionary and Theoretical Immunology, Department of Biology, University of New Mexico, MSC03 2020, Albuquerque, NM 87131, USA 2 Department of Biochemistry and Molecular Biology, University of New Mexico, MSC08 4670, Albuquerque, NM 87131, USA

Abstract The macromolecules contributed by the freshwater gastropod Biomphalaria glabrata, intermediate host of Schistosoma mansoni, to developing offspring inside egg masses are poorly known. SDS- PAGE fractionated egg mass fluids (EMF) of M line and BB02 B. glabrata were analyzed by MALDI-TOF (MS and tandem MS). A MASCOT database was assembled with EST data from B. glabrata and other molluscs to aid in sequence characterization. Of approximately 20 major EMF polypeptides, 16 were identified as defense-related, including protease inhibitors, a hemocyanin-like factor and tyrosinase (each with possible phenoloxidase activity), extracellular Cu-Zn SOD, two categories of C-type lectins, Gram negative bacteria-binding protein (GNBP), aplysianin/achacin- like protein, as well as versions of lipopolysaccharide binding protein/bacterial permeability increasing (LBP/BPI) that differed from those previously described from hemocytes. Along with two sequences that were encoded by “unknown” ESTs, EMF also yielded a compound containing a vWF domain that is likely involved in defense and a polypeptide with homology to the Aplysia pheromone temptin. Further study of B. glabrata pheromones is warranted as these could be useful in efforts to control these schistosome-transmitting snails. Several of the EMF polypeptides were contained in the albumen gland, the organ that produces most EMF. Thus parental investment of B. glabrata in immunoprotection of its offspring is indicated to be considerable.

Keywords Fresh water mollusc; proteomics; bioinformatics; Lipopolysaccharide binding protein/bactericidal permeability-increasing protein; C-type lectins; Cu-Zn SOD; pheromone

*CM Adema, CETI Biology MSC03 2020, 1 University of New Mexico, Albuquerque NM 87131-0001, ph +1 505 277 2743, fax +1 505 277 0304, [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Hathaway et al. Page 2

1. Introduction The fertilized eggs of many animal species undergo development within egg masses deposited NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript in the external environment, and the molecules used to protect the developing from attack by pathogens are of fundamental immunobiological interest. Using the freshwater Neotropical pulmonate snail Biomphalaria glabrata as our study organism, MALDI-TOF mass spectrometry was used to examine the polypeptide components of egg masses. Biomphalaria glabrata is an important intermediate hosts for the digenetic trematode Schistosoma mansoni, a parasite that causes a chronic, debilitating infection known as schistosomiasis that afflicts millions of people in developing countries [1]. A better underlying knowledge of the basic biology of the snail host will provide needed perspective for developing alternative and novel control strategies for disrupting the transmission of schistosomes by snails.

Several steps in the process of producing and depositing egg masses are vulnerable to exploitation by parasites and pathogens. Copulation may lead to venereally transmitted infections that impair host health or reproduction [2]. Pathogens of the reproductive system can also be passed on to the offspring via gametes or by being packaged in the fertilized eggs. Although these topics have not been well-studied in snails, venereal pathogen transmission is known in land snails both for a kinetoplastid [3] and nematodes [4]. Rickettsia-like organisms occur in molluscs and in their ova [5], and nematodes can colonize their host’s eggs, resulting in newborn snails that are already infected [6]. It is likely that snails employ immune protection of the reproductive tract against sexually-transmitted pathogens, and to prevent vertical transmission of pathogens. Such measures probably extend to the egg masses to protect the energetically expensive progeny.

Once deposited in the environment, developing egg masses face continuous threats from bacteria, fungi and other potential pathogens. Several studies report antimicrobial activity in the soluble components of molluscan egg masses [7–9]. The eggs of the marine opisthobranch gastropod Aplysia kurodai contain aplysianin-A, an L-amino acid oxidase (LAAO) which has antimicrobial activity against both Gram (+) and (−) bacteria. It is produced in the albumen gland and is part of the perivitelline fluid that envelops each egg [10]. Antimicrobial factors are also present in the eggs of pulmonate snails; the perivitelline fluid of Helix pomatia contains an N-acetylgalactosamine-binding lectin that agglutinates bacteria [11–13] and binds to herpesviruses [14]. The albumen gland of the freshwater pulmonate Lymnaea stagnalis produces an epidermal growth factor and a trypsin inhibitor, the latter postulated to prevent degradation of proteins in perivitelline fluid of the eggs [15].

Biomphalaria glabrata is a hermaphrodite like all pulmonate snails, and although capable of selfing, it is a preferential outcrosser [16]. In this snail species, oocytes produced in the ovotestis move via the hermaphroditic duct to the carrefour region where fertilization occurs. Eggs are then coated with perivitelline fluid which is synthesized and secreted by the albumen gland, a compound tubular exocrine gland that is associated with the female portion of the reproductive tract. Eggs are subsequently encapsulated by a membrane produced by the pars contorta. The packaged eggs are then surrounded by secretions of the muciparous and oothecal glands to form an egg mass which is deposited through the vaginal opening on smooth objects in the environment. Egg masses (Fig. 1) are flat, oval in shape, may exceed 1 cm in diameter, are of a transparent yellow-orange color, and contain 30 or more eggs [17].

The perivitelline fluid of B. glabrata contains the highly branched and proteins [18,19]. It is the major source of nutrition for fertilized eggs [20,21], and has agglutinating activity as do extracts of the AG [22–25]. Although the protein composition has not been systematically studied, Bai et al. [26–28] found phenoloxidase activity in both the AG and perivitelline fluid of B. glabrata, and associated the activity with SDS-PAGE protein

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band of 35 kDa. Miller et al. [29] recorded pBS11, a transcript in the albumen gland of B. glabrata that has 83% nucleotide identity to HdAGP, a major component of the albumen gland

NIH-PA Author Manuscript NIH-PA Author Manuscriptand perivitelline NIH-PA Author Manuscript fluid of the closely related snail, Helisoma duryi [30]. HdAGP is assumed to be a nutritive glycoprotein, yet it shares sequence similarity to lipopolysaccharide binding protein/bactericidal permeability-increasing (LBP/BPI) antimicrobial proteins [30]. A homolog of aplysianin-A, another antimicrobial peptide was found in whole bodies of B. glabrata [31] but its expression status in the AG is unknown. Proteomic approaches led to identification from B. glabrata of two isoforms of both a glycolytic (endo-1,4-β- mannanase 1 and 2) and a -binding protein, and an inhibitor of a cysteine protease (Bg type-2 cystatin), all of which were strongly expressed in the albumen gland [32]. Thus, the albumen gland produces perivitelline fluid and has a clear reproductive function and it is also implicated in having a significant role in the snail immune response [32]. Jeong et al. [24] speculated that the AG was the source of blood-borne agglutinins in B. glabrata. This supposition was supported by Guillou et al. [33] who identified transcripts encoding BgSel, a C-type lectin that was expressed in the peripheral secretory cells of the albumen gland and that was upregulated following exposure of resistant B. glabrata to the digenean parasite Echinostoma caproni.

To improve our understanding of both the reproductive biology and immunology of B. glabrata, we have applied MALDI-TOF techniques to characterize egg mass and albumen gland proteins from the BB02 and M line strains of B. glabrata. To facilitate our analysis, we recruited into MASCOT publicly available transcriptome sequence data for all representatives of the Mollusca, including B. glabrata. By emphasizing the acellular fluid of egg masses, this study also provides for B. glabrata more information on an important life cycle stage that may not yield easily to more conventional approaches based on EST analysis.

2. Materials and Methods 2.1. Egg mass sample collection Egg masses were collected from snails of the BB02 or M line strains of B. glabrata that are routinely maintained at the University of New Mexico [34,35]. Adult snails were housed in tanks containing artificial spring water and fed red leaf lettuce ad libitum. Pieces of styrofoam were floated on the water’s surface as the snails’ preferred egg laying substrate [26]. Egg masses deposited within 24–48 hours (Fig. 1) were removed from the styrofoam, rinsed in distilled water, blotted dry and placed in the lid of a sterile 1.7 ml tube, one egg mass per tube. A 22G needle was used to pierce each ovum, while avoiding disruption of the , and to release perivitelline fluid from the egg mass. The remaining solid portion of the egg mass including the embryos was discarded. The tube was closed and spun, the collected fluid is referred to as egg mass fluid (EMF). EMF consists primarily of perivitelline fluid but the possibility that it contains small amounts of fluid from the portion of the egg mass surrounding the individual membrane-bound ova can not be excluded. Forty μl of Laemmli sample buffer, prepared with 5% (v/v) BME according to instructions of the supplier (BioRad), but with increased SDS content of 20% (w/v), was added to the EMF. The sample was vortexed for 30 seconds, boiled for 5 minutes and then cold quenched.

2.2. Albumen gland protein extraction Albumen glands (AG) were dissected out of adult BB02 snails measuring 10–12 mm in shell diameter (Fig. 1). A single excised AG was immediately placed in a sterile 1.7 ml tube with 100 μl of Laemmli sample buffer (prepared as above), disrupted with a Kontes pestle (Kimble/ Kontes), boiled for 5 minutes and then cold quenched. The resulting sample was spun to remove any particulate debris.

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2.3. SDS-PAGE EMF or AG samples were run on a Bio-Rad Protean II system (Bio-Rad), connected to a VWR

NIH-PA Author Manuscript NIH-PA Author Manuscript1140 NIH-PA Author Manuscript cooling bath. The 1.5 mm thick gel consisted of a 4% acrylamide stacking gel (0.1% SDS in 0.125 M Tris-HCl pH 6.8) and a 5–20% gradient acrylamide separating gel (0.1% SDS in 0.375 M Tris-HCl pH 8.8). The protein samples were separated for ~3.5 hours at 70 mA until the dye front was approximately 2.5 cm from the bottom of the gel. Gels were rinsed and stained in Coomassie blue G250 (BioRad). Gels were photo-documented and sections containing prominent bands were excised.

2.4. In-gel peptide digest and mass spectrometry

Excised gel fragments were washed 3 times in 25 mM NH4HCO3/50% acetonitrile (ACN) for 20 minutes at 37°C. For in-gel digestion of proteins [36], gel pieces were incubated with 12.5 ng/μL trypsin in 25 mM NH4HCO3/10% ACN for 30 minutes at 37°C, then 25 mM NH4HCO3 was added to cover all of the gel and the samples were incubated overnight at 37° C. Five μL of peptide digest were bound to a C18 ZipTip (Millipore, Billerica, MA) and eluted in 2 replicate spots onto a MALDI plate with 5 mg/mL α-cyano-4-hydroxycinnamic acid in 50% ACN/0.1% trifluoric acid and allowed to air dry. Peptide spectra were acquired using a 4700 Proteomics Analyzer (TOF/TOF) (Applied Biosystems) at the Mass Spectrometry Facility, University of New Mexico (Albuquerque, NM). The instrument was in positive ion reflection mode. An MS survey scan was first performed for each sample across the entire plate and the 20 most intense peaks above a signal to noise ratio of 30 from each well were subjected to MS/MS analysis.

2.5. Custom MASCOT database for molluscan transcripts The MASCOT™ search engine software (Matrix Science) used to analyze MS data for protein identification relies on reference sequence information from a primary “MASCOT database” that encompasses the annotated proteins in the NCBI databases. The relative paucity of annotated protein sequences from molluscs limits the capabilities of MASCOT for meaningful analysis of MS data obtained from B. glabrata [32,37]. To increase the validity of MASCOT analysis, all publicly available ESTs from molluscs were downloaded from the respective GenBank and the Trace Archive databases at NCBI (http://www.ncbi.nlm.nih.gov/) in February, 2008. The majority of these sequences derived from Lottia gigantea (NCBI Taxonomy ID: 225164), Aplysia californica (NCBI Taxonomy ID: 6500), and B. glabrata (NCBI Taxonomy ID: 6526). All six reading frames of each EST were translated using Transeq (http://www.ebi.ac.uk/Tools/emboss/transeq/index.html). The resulting amino acid sequences (FASTA format) were placed in a primary sequence database “All Molluscs” (available upon request). MASCOT software performed in silico trypsin digests on all predicted amino acid sequences, creating fragment ion mass values for all protein sequences encoded for by molluscan (nucleotide-level) ESTs.

2.6. Database search and bioinformatic analysis Both MS and MS/MS spectra were processed using GPS Explorer™ software (v3.5, Applied Biosystems). For MS spectra a S/N ratio threshold of 30 was used and for MS/MS a threshold of 20 was used to detect peaks. Monoisotopic peak lists were generated in GPS Explorer™ and submitted to the MASCOT search engine for protein identity. Three databases were searched using MASCOT: NCBI non redundant, Swiss-Prot, and the locally created EST database “All Molluscs”. For all searches the precursor tolerance was 200ppm, the MS/MS fragment tolerance was 0.1 Da, and the variable modifications were carboxymethyl and oxidation. Protein identification was considered significant with a MASCOT score (C.I. %) corresponding to p<0.05. For all results with p<0.05 in the “All Molluscs” database, the corresponding original ESTs derived from B. glabrata were compiled and clustered if possible (Sequencher v4.8, Gene

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Codes). Assigned peptide sequences were checked to determine that they were encoded by open reading frames of originating ESTs. If identified by BLAST searches

NIH-PA Author Manuscript NIH-PA Author Manuscript(http://blast.ncbi.nlm.nih.gov/Blast.cgi), NIH-PA Author Manuscript complementary ESTs from B. glabrata were used to extend the coding sequences of the original matching ESTs, using Sequencher for clustering, requiring >95% nucleotide identity in the overlap. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the bioinformatics web tools at the Expasy site (http://www.expasy.ch/) were used for further sequence analysis, including predictions of signal peptide sequences and molecular weight of mature protein sequences.

3. Results 3.1. SDS-PAGE profiles of EMF and AG The SDS-PAGE separation of several replicate samples yielded consistent patterns both for EMF (from M line and BB02 strain B. glabrata) and for AG (BB02). Fewer protein bands were contained in EMF than in the more complex AG profile, but several bands of similar molecular weight were in common between the two types of samples. Figure 2 shows the gel sections and the protein bands from EMF and AG profiles that were excised for proteomic analysis.

3.2. Sequence identifications At a confidence level ≥95%, proteomics analyses yielded identification of 16 different polypeptides among the approximately 20 bands in Coomassie-stained lanes loaded with BB02 EMF. Thirteen of the 16 components were defense-related factors. Thirteen proteins were identified by examination of the EMF of M line B. glabrata, 9 of which were in common with those found in BB02 EMF, and 10 of which were defense-related. Among the putatively defense-related proteins identified in BB02 egg masses were α2-macroglobulin protease inhibitor, two serine-protease inhibitors (including ovalbumin), hemocyanin, LBP/BPI (several polypeptides ranging from 60 to 50 kDa; Fig. 2,3), two different categories of C-type lectins (see below), Gram-negative binding protein (GNBP), a vWF domain containing sequence, and a tyrosinase. An aplysianin/achacin homolog and extracellular superoxide dismutase (EC Cu/Zn SOD) were found from M line egg masses, as was a homolog of temptin, a pheromone described from the genus Aplysia, none of which was confidently recovered from BB02 egg masses. The corresponding analysis of solubilized BB02 AG identified 14 proteins at a confidence level ≥95%, nine of which were defense-related, nine of the 14 were also found in EMF. Other sequences recovered from AG were heat shock protein 90, elongation factor 2 (EF-2), ATP synthase subunit (mitochondrial) and ribosomal protein S8, as could be expected from a metabolically and transcriptionally active tissue. Finally, mass spectrometry data from other bands excised from SDS-PAGE profiles were linked to so-called “unknown” ESTs from B. glabrata that encode factors without similarity to known proteins. While their functions remain unknown, one such sequence occurred in AG, and two in EMF (Table 1).

3.3 Detailed consideration of C-type lectin sequences Several peptides obtained from EMF and AG matched to ESTs from B. glabrata that have a conserved domain (termed CLECT, C-type lectin (CTL)/C-type lectin-like (CTLD) domain, accession: cd00037; http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi), that is characteristic for C-type lectins. Two different categories of C-type lectins were recognized. Analysis of ESTs associated with 33–35 kDa polypeptides from EMF and AG, disclosed lectins that consisted of a carboxy-terminal C-type lectin domain and a putative N-terminal immunoglobulin superfamily (IgSF)-like domain. The latter domain has similarities with the IgSF domains of selectin and fibrinogen-related proteins (FREPs), lectins previously described from B. glabrata [38–40], (Fig. 4).

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Polypeptides of 16–18 kDa (present in both EMF and AG) represented members of a second category of C-type lectins, consisting of a single C-type lectin domain. Analysis yielded

NIH-PA Author Manuscript NIH-PA Author Manuscriptmultiple NIH-PA Author Manuscript B. glabrata-derived ESTs that encompass at least six related yet different lectin sequences of this latter category (Fig. 5).

4. Discussion SDS-PAGE in conjunction with proteomics analysis enabled identification of protein constituents of EMF and AG, and supports the notion that AG contributes proteins to the EMF. Without a large set of annotated proteins, de novo interpretation of MS spectra is challenging and may fail to identify all mass spectrometry data recovered [32,37]. Also, given that PVF is acellular, characterization of this distinctive product of the snail genome is refractory to EST- based approaches that monitor transcription. The recruitment of all unannotated transcriptome data from mollusks, including those from B. glabrata, improved the rate of identification of snail-specific polypeptide sequences. The use of the mass spectrometry-derived peptide fragments for BLAST analyses of the EST databases yielded (clusters of) transcribed sequences with open reading frames that provided additional information on the partial or complete amino acid sequence of the originating B. glabrata proteins (also see Fig. 3). This strategy will prove valuable for studying the proteome of other organs of B. glabrata, and for helping to interpret the forthcoming genome sequence [41].

The task of identifying proteins of EMF was aided by the fact that the total number of major bands in the SDS-PAGE profile was limited, such that proteins could be separated and obtained in relatively pure form. Seven major bands constituted large proportions of the total protein content. Possibly, some or all EMF proteins have a dual function, providing both nutrition and immune protection, at least during the early stages of embryological development, especially before immune cells are produced in the embryo. This is consistent with the hypothesis of Mukai et al [29] that the major 60–70 kDa component of egg masses of Helisoma duryi (and AG), that has extensive sequence similarity with to antimicrobial LBP/BPI proteins (also present in EMF and AG of B. glabrata, see below) and that diminishes over time with the development of the snail embryo’s, also has a nutritive function.

Among the defense-related polypeptides identified in BB02 egg masses was a ~175 kDa α2- macroglobulin. A tetrameric form of α2- macroglobulin with subunits of approximately 200 kDa has been recovered from B. glabrata plasma [42], and the peptides identified here match the amino acid sequence encoded by the full-length cDNA sequence (Genbank ACL00841) characterized from B. glabrata. These thio-ester-containing, broad spectrum protease inhibitors are important components of the innate defense systems of both invertebrates and vertebrates. They physically entrap pathogen-derived proteases and can also covalently bind other pathogen-derived ligands and modulate lectin-dependent cytolytic pathways [43,44]. A cysteine protease produced by S. mansoni larvae is inhibited by α2- macroglobulin in B. glabrata [45]. Along with putative defense functions, the protease inhibitory activity of α2- macroglobulin may protect the proteinaceous components of PVF from premature degradation, thus favoring the nutrition of the developing embryo.

Two serine protease inhibitors (serpins), including ovalbumen, are present in EMF. Like α2- macroglobulin, they may safeguard the protein components of the egg mass from “promiscuous proteolysis” [46]. Provision of egg masses with serpins is a likely defense strategy to protect against serine proteases that are major virulence factors for pathogens ranging from viruses to helminths [47]. Also serpins have important regulatory roles in enzymatic cascades such as that involved in melanization via the activation of phenoloxidase [48]. This is of interest given that phenoloxidase activity is present in the AG and egg masses of this species [26–28] and see below.

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Peptides with similarity to hemocyanin were recovered from a ~150 kDa band of EMF. As noted by Lieb et al. [49], planorbid snails like B. glabrata are unique among gastropods for

NIH-PA Author Manuscript NIH-PA Author Manuscripttheir NIH-PA Author Manuscript use of hemoglobin as a respiratory pigment. Nonetheless, hemocyanin is also present in B. glabrata blood but in a truncated form that makes it unlikely to function in oxygen transport [49]. Because of their sequence similarities to tyrosinases and phenoloxidases, possibly, the hemocyanin-like sequences in both the blood and egg masses may have tyrosinase activity and can stimulate the formation of melanin [50,51]. Although tyrosinase activity has yet to be documented for molluscan hemocyanins, the intrinsic phenoloxidase activity of hemocyanin from horseshoe crabs is activated by exposure to antimicrobial peptides [52]. Furthermore, hemocyanins themselves can also yield antimicrobial activities [53,54]. The involvement of hemocyanin (subunits) in defense against bacteria, fungi and viruses has also been highlighted in recent work with arthropods [55,56], and may help to explain why short forms of hemocyanin persist in planorbid snails.

As noted by Bai et al. [26], phenoloxidase activity is present in the AG and egg masses of B. glabrata. It was concluded that this activity is critical to the formation of the membrane around each individual egg which is akin to the tanned “egg shell” of other invertebrates. This phenoloxidase activity was associated with a 35 kDa protein from egg masses [27], much smaller than the ~150 kDa hemocyanin described above. However, also present in EMF are bands of 27–30 kDa with peptides similar to invertebrate tyrosinases, and these bands very likely correspond to the factor with phenoloxidase activity found by Bai et al. [27]. It is conceivable that two distinct sources within B. glabrata egg masses, hemocyanin and tyrosinase, contribute to melanization for egg membrane formation and for anti-pathogen functions.

Peptides from the most abundant protein component of EMF (at ~60kDA) enabled assembly of several ESTs (Fig. 3), the combined sequence of which displayed extensive similarity to a developmentally regulated albumen gland protein from B. glabrata that was later identified as LBP/BPI (lipopolysaccharide binding protein/bactericidal permeability-increasing protein) [29,33]. We confirmed that this anti-microbial molecule was prominently expressed in the AG of B. glabrata, and Mitta et al. [57] showed that LBP/BPI was down-regulated following exposure to trematode infection, whereas a related molecule expressed in hemocytes was upregulated following exposure to digeneans. This prompted the suggestion that B. glabrata has separate immune pathways for responding to bacteria and digeneans [33]. Our analysis of relevant ESTs indicated that multiple forms of LBP/BPI with minor sequence variation exist in EMF, all of which are distinct from the hemocyte version of the molecules reported by Mitta et al. [57] (represented by ESTs FC859212, FC855777, EE049679). The presence of variant forms is of interest in suggesting an enhanced spectrum of binding to potential microbial pathogens. A molecule with 83% sequence identity was also identified as the most abundant component of the PVF of the related snail, Helisoma duryi [30]. In larvae of the oyster Crassostrea gigas, LBP/BPI transcripts are upregulated following exposure to bacteria, and LBP/BPI may associate with the epithelium as part of first line of defense during all early stages of larval development [58]. LBP/BPI-related molecules have been proposed to be part of the first line of defense in the innate immune response of vertebrates as well [59].

The EMF contained Gram-negative bacteria-binding protein (GNBP), the peptides recovered from a 30kDa band all map to a previously characterized sequence of a 55kDa Biomphalaria glabrata GNBP (EF452345) [60]. This may indicate that GNBP is subject to cleavage in the EMF, or alternatively, the 30 kDa GNBP in EMF stems from as yet uncharacterized genes of B. glabrata. GNBPs are pattern recognition receptors that bind β-glucans of microbial cell walls and activate innate immunity through Toll/Imd pathways in arthropods [61] where GNBPs are also involved in melanotic encapsulations [62]. GNBPs are known from B. glabrata, and expression levels in whole-body extracts were either down-regulated following

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microbial infection or showed no change following digenean infection [60]. The locations of expression of GNBPs in B. glabrata are not known but this study also identified GNBP from

NIH-PA Author Manuscript NIH-PA Author Manuscripta 55kDa NIH-PA Author Manuscript band of the AG.

The same two distinct types of C-type lectins were found in both EMF and AG. Lectins are hypothesized to play a major role in non-self recognition in invertebrates including molluscs [63]. C-type lectins comprise a complex superfamily of calcium-dependent, - binding molecules that mediate a variety of defensive functions including agglutination and opsonization associated with phagocytosis [64]. The first type consisted of lectins (ranging from 33 to 35 kDa) that combine a downstream C-type lectin domain and upstream IgSF-like sequence, similar to the domain structure of selectin, another C-type lectin (dissimilar in sequence) described previously from B. glabrata [38]. Such a domain organization is also evident from FREPs, yet another group of B. glabrata lectins that feature N-terminal IgSF domains with fibrinogen-like sequences as downstream lectin domain [39,40]. It may be that this particular modular domain organization is a common theme for a subset of lectins in B. glabrata. Polypeptides of about 16–18 kDa represent the second type of C-type lectin that consists of a single C-type lectin domain with a signal peptide. Analysis identified six related yet dissimilar sequences of this type of lectin. One of the lectin sequences identified here (Genbank accession FC856131) has 99% identity (over 103 aa) with a partial transcript (EB709537) that is expressed in the peripheral secretory cells of the AG [33], additionally, expression was upregulated over four days following exposure to Echinostoma caproni in resistant snails. A closely related yet different sequence of this category of C-type lectins, not identified by this proteomics analysis, also exists in B. glabrata (Genbank accession EE049640). Using suppressive subtractive hybridization, Bouchut et al. [31] discovered that this C-type lectin was over-expressed in snails resistant to the digenean parasite Echinostoma caproni as compared to susceptible snails.

A superoxide dismutase of ~37 kDa was identified in the EMF of M line snails. This is an extracellular Copper/Zinc SOD (EC Cu/Zn SOD) that complements the two other dismutases described from Biomphalaria; the mitochondrial type Manganese (Mn) SOD [65] and a distinct cytoplasmic Cu/Zn SOD [66]. All SODs catalyze the dismutation of superoxide to hydrogen peroxide. The mitochondrial Mn SOD may play a role in a general stress response by limiting oxidative damage. Hydrogen peroxide produced by cytoplasmic Cu/Zn SOD in B. glabrata hemocytes kills sporocysts of S. mansoni and allelic SOD variants that achieve a high level of expression may be determinants of resistance to S. mansoni [66]. Guillou et al. [33] postulated a role for EC Cu/Zn SOD in limiting damage due to the generation of damaging radicals associated with encapsulation and killing of E. caproni sporocysts. The role of EC Cu/Zn SOD in EMF, whether to promote formation of hydrogen peroxide to discourage pathogens, or to protect the embryo from toxic oxygen radicals, remains to be determined.

Another immune-relevant protein identified in EMF from M line but not BB02 is a 55 kDa protein with similarities to achacin and aplysianin. Achacin is a bactericidal protein originally recovered from the body mucus of the terrestrial pulmonate snail Achatina fulica [67]. A related molecule is aplysianin-A, observed from eggs of Aplysia kurodai. It is produced in the AG, and has tumoricidal and bactericidal activity [68]. Achacin and aplysianin-A are L-amino acid oxidases (LAAO) that deaminate basic amino acids to produce hydrogen peroxide for killing of bacteria. The depletion of media of basic amino acids also inhibits bacterial growth [69]. Transcripts identical to those identified here were more abundant in echinostome resistant than susceptible B. glabrata [31].

Another putative defensive component identified in EMF and AG was a 50 kDa polypeptide that encodes a vWF domain such as contained in matrilin. Matrilin was expressed at increased

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levels in hemocytes from CB-R strain B. glabrata, selected for resistance to E. caproni, as compared to the susceptible EAF strain [70]. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript A final component of note identified in M line egg masses is a 17 kDa polypeptide with homology to temptin. In Aplysia, temptin is one of several peptide hormones produced in the AG and released into the environment with the eggs. Collectively this group of peptide hormones attracts conspecifics and stimulates formation of aggregations and hermaphroditic mating. This activity is mostly associated with attractin, but likely also involves other peptides including temptin [71]. Vianey-Liaud and Dussart [72] suggested that the fecundity of B. glabrata can be increased by exposure to a conspecific, even without physical contact, and even if sperm are not transferred, suggestive of a stimulatory chemical factor produced by the snail (see also [73] for a similar insight). Further study of the possible production and release of pheromones along with or in B. glabrata egg masses is warranted because it could offer some innovative approaches for developing pheromone traps to attract and eventually kill these schistosome-transmitting snails. Such traps might be used in selected transmission foci to minimize the number of snails available to transmit the infection.

There is much left to learn regarding the immunobiology of B. glabrata egg masses and of the developing embryos they contain. How do the components used to protect developing embryos differ from those used to protect eclosed snails, especially when the latter are exposed to different categories of pathogens? Do egg-protective measures change as a consequence of the infection history or status of the adult that produced the eggs? Some arthropod studies indicate that maternal pre-exposure to a pathogen can alter egg mass content in a way that reduces vulnerability of the offspring to the same pathogen [74]. Another fundamental question is whether defenses provided the eggs are entirely due to maternal provisioning. At what point do developing embryos contribute to their own defense? In the insect Manduca sexta, many maternally-produced immune-related proteins and corresponding mRNAs were detected in naïve eggs, yet an upregulation of anti-microbial peptide expression was noted upon challenge of eggs with bacteria, with the synthesis occurring in extra-embryonic tissues [75]. These authors postulated that extra-embryonic immunity may be a common mechanism for protecting early stage embryos because 1) an immune response mounted by the embryo may damage it or compromise its ability to develop properly; and 2) it would be advantageous to kill microbes in an extra-embryonic environment before they have the opportunity to colonize and damage the embryos [75].

By identifying several of the polypeptides present in EMF and the AG, our study will help answer these questions. This study is also of value in showing that proteomics approaches can help reveal the composition of acellular compartments refractory to transcriptomics studies (snail mucus is another logical target for such study), identify the AG as the likely source organ of several components of EMF, reveal important aspects of snail biology that may be relevant for control such as the possible production of B. glabrata pheromones, and when used in an organ specific context in conjunction with transcriptomics, will be very useful in ascribing functions to otherwise uncharacterized genes once the genome sequence of B. glabrata becomes available.

Acknowledgments

Bioinformatics support was provided by George Rosenberg from the University of New Mexico’s Molecular Biology Facility which is supported by NIH Grant Number 1P20RR18754 from the Institute Development Award (IDeA) Program of the National Center for Research Resources. NIH support is acknowledged by C. M. Adema (AI052363) and E. S. Loker (AI024340).

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Abbreviations/Symbols

NIH-PA Author Manuscript NIH-PA Author ManuscriptACN NIH-PA Author Manuscript acetonitrile Cu-Zn SOD Copper-Zinc superoxide dismutase Da Dalton GNBP Gram negative bacteria-binding protein IgSF immunoglobulin superfamily MS mass spectrometry MS/MS tandem mass spectrometry MALDI-TOF matrix assisted laser desorption/ionization- time of flight LAAO L-amino acid oxidase LBP/BPI lipopolysaccharide binding protein/bacterial permeability increasing (protein) vWF van Willebrand Factor

References 1. Gryseels B, Polman K, Clerinx J, Kestens L. Human schistosomiasis. Lancet 2006;368:1106–1118. [PubMed: 16997665] 2. Poiani A. Complexity of seminal fluid: a review. Behav Ecol Sociobiol 2006;60:289–310. 3. Current WL. Cryptobia sp. in the snail Triadopsis multilineata (Say): fine structure of attached flagellates and their mode of attachment to the spermatheca. J Protozool 1980;27:278–287. 4. Morand S, Faliex E. Study on the life cycle of a sexually transmitted nematode parasite of a terrestrial snail. J Parasitol 1994;80:1049–1052. [PubMed: 7799151] 5. Fries CR, Grant DM. Rickettsiae in gill epithelial cells of the hard clam, Mercenaria mercenaria. J Invertebr Pathol 1991;57:166–171. [PubMed: 2022867] 6. Anderson RC. On the development and transmission of Cosmocercoides dukae of terrestrial mollusks in Ontario. Can J Zool 1960;38:801–825. 7. Benkendorff K, Davis AR, Bremner JB. Chemical defense in the egg masses of benthic invertebrates: an assessment of antibacterial activity in 39 mollusks and 4 polychaetes. J Invertebr Pathol 2001;78:109–118. [PubMed: 11812113] 8. Benkendorff K, Pillai R, Bremner JB. 2,4,5-Tribromo-1H-imidazole in the egg masses of three muricid molluscs. Nat Prod Res 2004;18:427–431. [PubMed: 15248610] 9. Ramasamy MS, Murugan A. Fouling deterrent chemical defence in three muricid gastropod egg masses from the Southeast coast of India. Biofouling 2007;27:259–265. [PubMed: 17653935] 10. Takamatsu N, Shiba T, Muramoto K, Kamiya H. Molecular cloning of the defense factor in the albumen gland of the sea hare Aplysia kurodai. FEBS Lett 1995;377:373–376. [PubMed: 8549758] 11. Kőhler W, Prokop O, Kűhnemund O. Routine identification of group-C streptococci by means of an agglutinin (protectin) from the albumen gland of the edible snail, Helix pomatia. J Med Microbiol 1973;6:127–130. [PubMed: 4632252] 12. Patchett RA, Kelly AF, Kroll RG. The adsorption of bacteria to immobilized lectins. J Appl Bacteriol 1991;71:277–284. [PubMed: 1955420] 13. Sanchez JF, Lescar J, Chazalet V, Audfray A, Gagnon J, Alvarez R, Breton C, Imberty A, Mitchell EP. Biochemical and structural analysis of Helix pomatia agglutinin. A hexameric lectin with a novel fold. J Biol Chem 2006;281:20171–20180. [PubMed: 16704980] 14. Slifkin M, Cumbie R. Rapid detection of herpes simplex virus with fluorescein-labeled Helix pomatia lectin. J Clin Microbiol 1989;27:1036–1039. [PubMed: 2545739] 15. Nagle GT, de Jong-Brink M, Painter SD, Li KW. Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea. Eur J Biochem 2001;268:1213–1221. [PubMed: 11231272]

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 11

16. Paraense WL. Self- and cross-fertilization in Australorbis glabratus. Mem Inst Oswaldo Cruz 1955;63:285–291.

NIH-PA Author Manuscript NIH-PA Author Manuscript17. Sturrock, NIH-PA Author Manuscript RF. The intermediate hosts and host-parasite relationships. In: Jordan, P.; Webbe, G.; Sturrock, RF., editors. Human Schistosomiasis. Wallingford: CAB International; 1993. p. 33-85. 18. Geraerts, PM.; Joosse, J. Freshwater snails (Basommatophora). In: Tompa, AS.; Verdonk, NH.; van den Biggelaar, JAM., editors. The Mollusca 7: Reproduction. New York: Academic Press; 1984. p. 141-207. 19. Morishita F, Mukai ST, Saleuddin AS. Release of proteins and from the albumen gland of the freshwater snail Helisoma duryi: effect of cAMP and brain extracts. J Comp Physiol A 1998;182:817–825. [PubMed: 9631557] 20. Plesch B, de Jong-Brink m, Boer HH. Histological and histochemical observations on the reproductive tract of the hermaphroditic pond snail Lymaea stagnalis (L). Neth J Zool 1971;21:180–201. 21. Goudsmit EM. Galactogen catabolism by embryos of the freshwater snails, Bulimnaea megasoma and Lymnaea stagnalis. Comp Biochem Physiol B 1976;53:439–442. [PubMed: 4280] 22. Michelson EH, Dubois L. Agglutinins and lysins in the molluscan family Planorbidae: a survey of hemolymph, egg-masses, and albumen-gland extracts. Biol Bull 1977;153:219–227. [PubMed: 560878] 23. Stein PC, Basch PF. Purification and binding properties of hemagglutinin from Biomphalaria glabrata. J Invertebr Pathol 1979;33:10–18. [PubMed: 438526] 24. Jeong KH, Sussman S, Rosen SD, Lie KJ, Heyneman D. Distribution and variation of hemagglutinating activity in the hemolymph of Biomphalaria glabrata. J Invert Pathol 1981;38:256– 263. 25. Boswell CA, Bayne CJ. Isolation, characterization and functional assessment of a hemagglutinin from the plasma of Biomphalaria glabrata, intermediate host of Schistosoma mansoni. Dev Comp Immunol 1984;8:559–568. [PubMed: 6500135] 26. Bai G, Li J, Christensen BM, Yoshino TP. Phenoloxidase activity in the reproductive system and egg masses of the pulmonate gastropod, Biomphalaria glabrata. Comp Biochem Physiol B Biochem Mol Biol 1996;114:353–359. [PubMed: 8840512] 27. Bai G, Brown JF, Watson C, Yoshino TP. Isolation and characterization of phenoloxidase from egg masses of the gastropod mollusc, Biomphalaria glabrata. Comp Biochem Physiol B Biochem Mol Biol 1997;118:463–469. [PubMed: 9440238] 28. Bai G, Johnston LA, Watson CO, Yoshino TP. Phenoloxidase activity in the reproductive system of Biomphalaria glabrata: role in egg production and effect of schistosome infection. J Parasitol 1997;83:852–858. [PubMed: 9379290] 29. Miller AN, Ofori K, Lewis F, Knight M. Schistosoma mansoni: use of a subtractive cloning strategy to search for RFLPs in parasite-resistant Biomphalaria glabrata. Exp Parasitol 1996;84:420–428. [PubMed: 8948331] 30. Mukai ST, Hoque T, Morishita F, Saleuddin ASM. Cloning and characterization of a candidate nutritive glycoprotein from the albumen gland of the freshwater snail Helisoma duryi (Mollusca, Pulmonata). Invert Biol 2004;123:82–91. 31. Bouchut A, Coustau C, Gourbal B, Mitta G. Compatibility in the Biomphalaria glabrata/Echinostoma caproni model: new candidate genes evidenced by a suppressive subtractive hybridization approach. Parasitology 2007;134:575–588. [PubMed: 17096871] 32. Vergote D, Bouchut A, Sautière PE, Roger E, Galinier R, Rognon A, Coustau C, Salzet M, Mitta G. Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni. Int J Parasitol 2005;35:215–224. [PubMed: 15710442] 33. Guillou F, Mitta G, Galinier R, Coustau C. Identification and expression of gene transcripts generated during an anti-parasitic response in Biomphalaria glabrata. Dev Comp Immunol 2007;31:657–671. [PubMed: 17166585] 34. Adema CM, Luo MZ, Hanelt B, Hertel LA, Marshall JJ, Zhang SM, DeJong RJ, Kim HR, Kudrna D, Wing RA, Soderlund C, Knight M, Lewis FA, Caldeira RL, Jannotti-Passos LK, Carvalho Odos S, Loker ES. A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni. Mem Inst Oswaldo Cruz 2006;101 (Suppl 1):167–177. [PubMed: 17308766]

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 12

35. Loker ES, Hertel LA. Alterations in Biomphalaria glabrata plasma induced by infection with the digenetic trematode Echinostoma paraensei. J Parasitol 1987;73:503–513. [PubMed: 3598800]

NIH-PA Author Manuscript NIH-PA Author Manuscript36. Shevchenko NIH-PA Author Manuscript A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996;68:850–858. [PubMed: 8779443] 37. Jannotti-Passos LK, Andrade HM, Caldeira RL, Romanha AJ, Murta SM, Chapeaurouge DA, Perales J, Coelho PM, Carvalho OS. Proteome analysis of the cardiac and pericardial tissue of Biomphalaria tenagophila populations susceptible and resistant to Schistosoma mansoni infection. Acta Trop 2008;105:229–234. [PubMed: 18262165] 38. Guillou F, Mitta G, Dissous C, Pierce R, Coustau C. Use of individual polymorphism to validate potential functional markers: case of a candidate lectin (BgSel) differentially expressed in susceptible and resistant strains of Biomphalaria glabrata. Comp Biochem Physiol B Biochem Mol Biol 2004;138:175–181. [PubMed: 15193273] 39. Léonard PM, Adema CM, Zhang S-M, Loker ES. Structure of two FREP genes that encode IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata. Gene 2001;269:155–165. [PubMed: 11376947] 40. Zhang S-M, Léonard PM, Adema CM, Loker ES. Parasite responsive IgSF members in the snail Biomphalaria glabrata: characterization of novel genes with tandemly arranged IgSF domains and a fibrinogen domain. Immunogenetics 2001;53:684–694. [PubMed: 11797103] 41. Raghavan N, Knight M. The snail (Biomphalaria glabrata) genome project. Trends Parasitol 2006;22:148–151. [PubMed: 16497557] 42. Bender RC, Bayne CJ. Purification and characterization of a tetrameric alpha-macroglobulin proteinase inhibitor from the gastropod mollusc Biomphalaria glabrata. Biochem J 1996;316:893– 900. [PubMed: 8670168] 43. Armstrong PB, Quigley JP. Alpha2-macroglobulin: an evolutionarily conserved arm of the innate immune system. Dev Comp Immunol 1999;23:375–390. [PubMed: 10426429] 44. Padhi A, Buchheim MA, Verghese B. Dynamic evolutionary pattern of alpha2-macroglobulin in a model organism, the zebrafish (Danio rerio). Mol Immunol 2008;45:3312–3318. [PubMed: 18485482] 45. Fryer SE, Bender RC, Bayne CJ. Inhibition of cysteine proteinase from Schistosoma mansoni larvae by alpha-macroglobulin from the plasma of Biomphalaria glabrata. J Parasitol 1996;82:343–347. [PubMed: 8604113] 46. Silverman GA, Whisstock JC, Askew DJ, Pak SC, Luke CJ, Cataltepe S, Irving JA, Bird PI. Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis. Cell Mol Life Sci 2004;61:301–325. [PubMed: 14770295] 47. Armstrong PB. Proteases and protease inhibitors: a balance of activities in host-pathogen interaction. Immunobiology 2006;211:263–281. [PubMed: 16697919] 48. Kanost MR, Jiang H, Yu XQ. Innate immune responses of a lepidopteran insect, Manduca sexta. Immunol Rev 2004;198:97–105. [PubMed: 15199957] 49. Lieb B, Dimitrova K, Kang HS, Braun S, Gebauer W, Martin A, Hanelt B, Saenz SA, Adema CM, Markl J. Red blood with blue-blood ancestry: intriguing structure of a snail hemoglobin. Proc Natl Acad Sci USA 2006;103:12011–12016. [PubMed: 16877545] 50. Decker H, Jaenicke E. Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins. Dev Comp Immunol 2004;28:673–87. [PubMed: 15043938] 51. Decker H, Schweikardt T, Nillius D, Salzbrunn U, Jaenicke E, Tuczek F. Similar enzyme activation and catalysis in hemocyanins and tyrosinases. Gene 2007;398:183–191. [PubMed: 17566671] 52. Nagai T, Osaki T, Kawabata S. Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. J Biol Chem 2001;276:27166–27170. [PubMed: 11375396] 53. Destoumieux-Garzón D, Saulnier D, Garnier J, Jouffrey C, Bulet P, Bachère E. Crustacean immunity. Antifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge. J Biol Chem 2001;276:47070–47077. [PubMed: 11598107] 54. Lee SY, Lee BL, Söderhäll K. Processing of crayfish hemocyanin subunits into phenoloxidase. Biochem Biophys Res Commun 2004;322:490–496. [PubMed: 15325257]

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 13

55. Lei K, Li F, Zhang M, Yang H, Luo T, Xu X. Difference between hemocyanin subunits from shrimp Penaeus japonicus in anti-WSSV defense. Dev Comp Immunol 2008;32:808–813. [PubMed: 18234332] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 56. Jaenicke E, Fraune S, May S, Irmak P, Augustin R, Meesters C, Decker H, Zimmer M. Is activated hemocyanin instead of phenoloxidase involved in immune response in woodlice? Dev Comp Immunol 2009;33:1055–1063. [PubMed: 19447131] 57. Mitta G, Galinier R, Tisseyre P, Allienne JF, Girerd-Chambaz Y, Guillou F, Bouchut A, Coustau C. Gene discovery and expression analysis of immune-relevant genes from Biomphalaria glabrata hemocytes. Dev Comp Immunol 2005;29:393–407. [PubMed: 15707661] 58. Tirapé A, Bacque C, Brizard R, Vandenbulcke F, Boulo V. Expression of immune-related genes in the oyster Crassostrea gigas during ontogenesis. Dev Comp Immunol 2007;31:859–873. [PubMed: 17337052] 59. Levy O. Antimicrobial proteins and peptides of blood: templates for novel antimicrobial agents. Blood 2000;96:2664–2672. [PubMed: 11023496] 60. Zhang SM, Zeng Y, Loker ES. Characterization of immune genes from the schistosome host snail Biomphalaria glabrata that encode peptidoglycan recognition proteins and gram-negative bacteria binding protein. Immunogenetics 2007;59:883–898. [PubMed: 17805526] 61. Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu Rev Immunol 2007;25:697–743. [PubMed: 17201680] 62. Wang X, Fuchs JF, Infanger LC, Rocheleau TA, Hillyer JF, Chen CC, Christensen BM. Mosquito innate immunity: involvement of beta 1,3-glucan recognition protein in melanotic encapsulation immune responses in Armigeres subalbatus. Mol Biochem Parasitol 2005;139:65–73. [PubMed: 15610820] 63. Horak P, van der Knaap WPW. Lectins in snail - trematode immune interactions: a review. Folia Parasitologica 1997;44:161–172. 64. Bianchet, MA.; Ahmed, H.; Vasta, GR.; Amzel, LM. Structural aspects of lectin-ligand interactions. In: Vasta, GR.; Ahmed, H., editors. Animal Lectins: A Functional View. Boca Raton FL: CRC Press Taylor & Francis; 2009. p. 475-491. 65. Jung Y, Nowak S, Hertel LA, Zhang S-M, Loker ES, Adema CM. Manganese superoxide dismutase from Biomphalaria glabrata. J Invertebr Pathol 2005;90:59–63. [PubMed: 16081093] 66. Bayne CJ. Successful parasitism of vector snail Biomphalaria glabrata by the human blood fluke (trematode) Schistosoma mansoni: a 2009 assessment. Mol Biochem Parasitol 2009;165:8–18. [PubMed: 19393158] 67. Obara K, Otsuka-Fuchino H, Sattayasai N, Nonomura Y, Tsuchiya T, Tamiya T. Molecular cloning of the antibacterial protein of the giant African snail, Achatina fulica Férussac. Eur J Biochem 1992;209:1–6. [PubMed: 1396687] 68. Takamatsu N, Shiba T, Muramoto K, Kamiya H. Molecular cloning of the defense factor in the albumen gland of the sea hare Aplysia kurodai. FEBS Lett 1995;377:373–376. [PubMed: 8549758] 69. Jimbo MF, Nakanishi R, Sakai K, Muramoto, Kamiya H. Characterization of L-amino acid oxidase and antimicrobial activity of aplysianin A, a sea hare-derived antitumor-antimicrobial protein. Fish Sci 2003;69:1240–1246. 70. Bouchut A, Roger E, Coustau C, Gourbal B, Mitta G. Compatibility in the Biomphalaria glabrata/ Echinostoma caproni model: potential involvement of adhesion genes. Int J Parasitol 2006;36:175– 184. [PubMed: 16310790] 71. Cummins SF, Nichols AE, Schein CH, Nagle GT. Newly identified water-borne protein pheromones interact with attractin to stimulate mate attraction in Aplysia. Peptides 2006;27:597–606. [PubMed: 16309784] 72. Vianey-Liaud M, Dussart G. Aspects of pairing and reproduction in the hermaphrodite freshwater snail Biomphalaria glabrata (Gastropoda: Pulmonata). J of Moll Stud 2002;68:245–250. 73. Boyle JP, Yoshino TP. Monoamines in the albumen gland, plasma, and central nervous system of the snail Biomphalaria glabrata during egg-laying. Comp Biochem Physiol A Mol Integr Physiol 2002;132:411–422. [PubMed: 12020657] 74. Little TJ, O’Connor B, Colegrave N, Watt K, Read AF. Maternal transfer of strain-specific immunity in an invertebrate. Curr Biol 2003;13:489–492. [PubMed: 12646131]

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 14

75. Gorman MJ, Kankanala P, Kanost MR. Bacterial challenge stimulates innate immune responses in extra-embryonic tissues of tobacco hornworm eggs. Insect Mol Biol 2004;13:19–24. [PubMed: 14728663] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

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Fig. 1. (A) An egg mass of Biomphalaria glabrata. The raft (R) of this egg mass, between 24 to 48 hours old, contains 33 eggs (e). While taking care to not damage developing embryo’s (de), each egg was punctured with a 22G needle to release egg mass fluid (EMF). Remaining solids were discarded. (B) The white oval indicates the location of the albumen gland on the anatomy of B. glabrata with the shell intact (left) and for the same snail after removal of the shell (right). HF = headfoot, M = mouth, I = intestine, H = (location of) heart, AG =albumen gland, S = stomach, HP = hepatopancreas, OT = ovotestis.

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Fig. 2. Representative SDS-PAGE profiles of protein components of egg mass fluid and albumen gland of Biomphalaria glabrata Note bands of similar molecular weight between egg mass fluid (EMF) collected from one egg mass of both M line and BB02 strain B. glabrata and from the albumen gland of a single BB02 B. glabrata (AG). Letters and numbers to the left of each gel profile indicate the regions of gels that were excised for proteomics analysis. Protein identifications are indicated next to the gel lanes (also see table 1). The left two samples were separated on the same gel, the right SD- PAGE profile originated from another gel. Position of molecular weight markers (kiloDaltons) is indicated with bars to the left of each gel lane.

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Fig. 3. Matching MS peptide sequence data with ESTs from Biomphalaria glabrata Peptides recorded by mass spectrometry were matched with encoding B. glabrata ESTs in the MASCOT database. If BLASTN searches yielded additional ESTs or cDNAs with sequence similarities, these were clustered to generate extended coding sequences (boxes). Regions of matching peptide and encoding nucleotide sequence are indicated as hatched boxes, untranslated terminal regions are shown as single lines, SP = signal peptide, * = stopcodon, AAAA = poly-A tail. Lines show the relative positions of clustered ESTs along the complete cDNAs (≥95% sequence identity in overlap). (A) This EST cluster encodes for LBP/BPI, an antimicrobial factor, derived from the 60kDA protein band of both EMF and AG. The complete coding sequence is spanned by two ESTs (Genbank accessions FC857161 and FC857344), but the transcript has been captured by multiple ESTs of B. glabrata. The alignment excludes many other LBP/BPI-like ESTs from B. glabrata, suggesting that several sequence variants of LBP/BPI exist in B. glabrata. Likely, these contribute the similar yet variant LBP/BPI sequences from SDS-PAGE bands ranging from 60 to 50 kDa from both EMF and AG samples. Remarkably, all these variant sequences are distinct from the hemocyte version of LBP/BPI reported by Mitta et al. [60] (for which a complete coding sequence is represented by ESTs FC859212, FC855777, EE049679). (B) A 15 kDa band from EMF (BB02 and M line B. glabrata) contained peptide sequences that were encoded by unknown ESTs (representative EST FC859944). Transcripts were abundantly recovered from B. glabrata, but no functional context exists for the encoded sequence.

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Fig. 4. C-type lectins with N-terminal IgSF-like domains (A) Both albumen gland and egg mass fluids of B. glabrata contained two different C-type lectins that display upstream immunoglobulin superfamily (IgSF)-like domains. Peptide sequences from the 35 kDa bands mapped to a cluster of three ESTs (Genbank accessions DW474380, FC856550, FC859543) that contained a complete coding sequence. This lectin is designated “34kDa” based on the predicted molecular weight for the mature protein sequence. The protein bands of 35 and 33 kDa yielded identical MS peptide data for another lectin (designated 3335k), however, the sequence represented by ESTs FC857569 and EE049674 remains incomplete at the N-terminus. The alignment compares the deduced amino acid sequences. Shaded regions represent experimentally recovered amino acid sequences. The signal peptide is within a solid line box, dotted line boxes locate cysteine residues that form disulfide bonds to create the intrachain loop that is typical for IgSF domains. The C-type lectin conserved domains (NCBI CCD cd00037) are underlined, + = conserved replacements. (B,C) The upstream IgSF domains of the 34kDa and 3435k lectins display similarities (identical residues and conserved replacements) to the IgSF domains of selectin (BgSel; AAC14140) and fibrinogen-related protein 4 (BgMF4; AAC47699), two previously characterized lectins from B. glabrata, respectively.

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Fig. 5. Single domain C-type lectins Peptide information from two low molecular weight protein components of albumen gland and egg mass fluids of B. glabrata revealed a group of similar but distinct C-type lectins that range from in 16 to 18 kDa (predicted molecular weight of mature proteins). The alignment shows representative EST sequences (identified by Genbank accession number) that each encode for a signal peptide (box) and a conserved C-type lectin domain (NCBI CCD cd00037; underlined). Shading denotes experimentally recovered peptide sequence data. Some MS polypeptide sequence fragments were obtained only from either EMF or AG samples but the extensive sequence identity among these lectins prevents distinction of tissue specific transcripts. The alignment shows identical and conservedly replaced amino acids of the most divergent sequence FC856131 (18.053 kDa) relative to remaining highly similar lectins, FC857919 (16.260 kDa); FC859813 (16.259 kDa); FC859217 (16.260 kDa) and FC859582 (18.367 kDa). Unique amino acid replacements within the latter group are indicated as white letters on black fields.

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 20 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript , similar to that of ABR68007 Lehmannia valentiana Aplysia kurodai ACL00841 thioester- containing protein Biomphalaria glabrata Biomphalaria glabrata Achatina fulica Taeniopygia guttata CAG28309 Callicebus moloch AAB00448, AAB00448, Megathura crenulata matrilin-like 40 kDa protein ID with significant BLAST similarity (bold recovered from EMF and AG) Alpha-2-macroglobulin Biomphalaria glabrata Hemocyanin Serine proteinase inhibitor, (ovalbumin) ACA57865 Angiotensin-converting enzyme (ACE) XP_002191587 LBP/BPI, LBP/BPI, achacin/aplysianin-like CAA45871 achacin, BAA11867 aplysianin-A, vWF domain containing sequence d TABLE 1 and identification of polypeptides. representative coding sequences EV818539 ES748875, CN655026, CN779709 ES485607 FC856582 EE049678 CK640663 FC859131 EE049678 FC857472 EE049679 FC858901 CV042560 FC856487 Biomphalaria glabrata c peptide sequences recovered SYSMSNNYLQLSLLSK, AESDVLIK, ITSTEAIDSLAYEIR, VEPSWAPIAQLLMYYIR AEIHATLHTGER, FAVLGGPTELGWR, NIQELTDGESNNLR, LLTVQFEQALSR, LQSETSADNFENIAGFHFAPNR, EWTDREVNLK, CPPHGSDRFACSPHGLPIFPHWHR, LGASWGIPYWDWTDESTALPK, LFSDPEDNPFYR, LWTMWQQLQYLR, HVDSSMEPFR NYIVVPAYFMR, RLGFFQTK, FPLKNPR, FSFYIVTSADMPK, LEHTIAR, GNAQFFR GWSDASGR, DYQLLLDVWK, KPTFDVTDEMIR, LFDXSMALESVSR VREWTEERLCR, LSAEMDEFKVDYSPVSK, VDYSPVSKVSVSQQSLEAR, FTHMVVR, SDSTPSSQKPDDLPR, TFAESAHSHDYLK, EDQDDEDKMFNLWLDEFVAK, EVSSSKVPDVEISEEGVR, HRGEVSWK, KGLPLMSLEDLSLLDPTVR, VQLHGDVLVSTR, LSYVSSLIPELSSNSAGSVQVEVSSSK, LIPDLLSYDVETSELTAIK, VITDYIDNK, MFNLWLDEFVAK VREWTEERLCR, LSAEMDEFKVDYSPVSK, VSVSQQSLEAR, FTHMIVR, TFAESAHSHDYLK GEVSWKSDSTPSSQKPDDLPR, EDQDDEDKMFNLWLDEFVAK, SVQVEVSSSKVPDVEISEEGVR, HRGEVSWK KGLPLMSLEDLSLLDPTVR, VQLHGDVLVSTR LSYASSLIPELSSNSPDSYLQVEVSSNK, MFNLWLDEFVAK, IAEDTISEQDQR, LIPDLLSYDVETSELTAIK, INPKEADAFK IIQWEPLK, ELFDYIMR, SSLVSEALDSVR, AYVLQASFAEGDR, SVPVSTVVMTFSQR, ASVLFTDESISQVVELGQSPDSR GIPNNDAPKACK, FGLVIFSDNAR, TPYLLGNTFTDK, NLQTLLYVTAQK, SPAHVFTTDEYK, ALVMANSLLATTGRPGVPK, IFDLSTYNNHPELLEAILK, FTLGVNDVLFAEIIYGAVVQK TLADIIFVLDSSGSIGADNYIK, KIFDLSTYNNHPELLEAILK, VVITLTDGNSANAMLTFGAATALK b MW 175 150 100 100 60 55 55 50 a B,11) D,13) E,14) ( ( ( 12) 14) A) C) M) ( ( ( ( ( Sample B B,M B M B,M B,M M B Proteomics data obtained from EMF and AG of

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 21 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Biomphalaria Biomphalaria glabrata Manduca sexta Haliotis discus contain IgSF-like and conserved contain conserved C-type lectin -like domain (CTLD; , contains IgSF-like and conserved AAB00448, C-type lectin-like (CTLD; NCBI CDD cd00037) domains glabrata ID with significant BLAST similarity (bold recovered from EMF and AG) LBP/BPI, triacylglycerol lipase, LOC657679 Tribolium castaneum Serpin 1 AAC47333 Cu/Zn Superoxide dismutase ABL75447 Hypocrea lixii C-type lectin C-type lectins, EF452345, Gram-negative bacteria binding protein NP_492055 TYRosinase family member (tyr-3) Caenorhabditis elegans Unknown temptin, ABO26633 C-type lectins C- type lectin- like (CTLD; NCBI CDD cd00037) domains NCBI CDD cd00037) d representative coding sequences EE049679 EV821304, EV817471, EE723470 EX002351 EE049528 FC856550 FC857569 EF452345 ES272486 ES745519 ES486406 FC857191 FC856131, FC859813 c peptide sequences recovered FTHMIVR, RGLPLMSLPDLR VPDVEISEEGVR, IAEDTISEQDQR, LVPDILSYDVETR ESPSQSIDLPAIR, VDNINDVWTGLK, FIVHGFLEDTDKDSWLR, VVGAQIAHLIQK, ITGLDPAGPYFENTDIR, GIIEVNVATYSGVIK, FVDVIHTDGR, DILELGMR, SVKYFIESVNVK, CPYTAYPCSGEEDFVSGK, EAAREGACNHAR, LDPSDARFVDVIHTDGR TDDVNKFDVAEIAFTGSR, AFSDTLADFAGLTQK VDELFTGHGSYNVQMAIRKFK, AVIEVKESGTVAAAVTAADMFER HVGDYGNIELMTK, VTDNLSSLFGDNSILGR, SLVIHEFEDDLGVK, TEGTSGRIVSCCVIALPR TQFIISDSFR, TYFFTPNDAHFR, YFNWTIGQPDAGR, MWGDYTTYLQEK KSLVIECR, VEACYEINYFNIIR, YIEISSDFAGVVGR, DAVLTGSTSDTMSAFLK, TDEYKPQYDK, QGLVDLFFLK, ATGSLISGTDAEKER, TWLYHR, EMTYFNWLKPAPDNLQGK LMSNPTLR, YGVVEVR, ISKGDWIWPAIWMLPR, GNEVGIGISQVSSTLHWGPSPNENR, TGSWYDSFHTWR, GGFGGQNIWAGGER, KPWANNAPAPDVDFFNAR, NEWLPTWR, GDETAMIVDYVEFR, GRLVTFFLHEYFK, IVASATGYDRK, DNVYGGWPR, DDFNYIDTNNWR, YEVSAYGGLNR, EFQVYTNDPR, SGNLYLKPTISTDDPR, FNENTLR, WGVMDVAR TTYEPVPTCSAR, LWTPDFFGTFR, GPVVDGPFAGWR, LPTGVQLIR, NAGVDGDLLTPQIISNILTR, SIGVNPAQYPR, HEPTATTGHGSUTQEYGYSDALASR, TTYEPVPTCSAR, VPDTCNVERNYAIPQ, SPTCGVGSLVCEVSTGR, QSSDKVPDT, NYAIPNQNDYCCEDK LRGDLVR, MIINVDR, GMSTVALSK, IGLLSDQVMR ILLYVSTWDK, DTLLETFNFVR, WYPYSQEYMK, AGLALGQGAAFGNAAALAANLGLAK, SAPILLPPLKDTLLETFNFVR EQLFMMK, WHGVGHTNPSGGLAR, AANFTWTK, TTDITHPGINPK ILFQQALLNK, LYYASKDTVANFDK, SQLTWVFQR, MSVFDWIPGEPNDK, b MW 50 45 45 35 34 35 33 30 30–27 18 18 18–15 a M,14) I, J,17) K,18) K,L,18) ( ( ( ( 15) 18) F) F) G) I) ( ( ( ( ( ( G,15) H,16) Sample B,M B B M B B,M ( ( B B,M B,M M B,M

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 22 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript β , similar to that of ABR68007 Biomphalaria glabrata Biomphalaria glabrata Xenopus (Silurana) tropicalis CAD88977 Modiolus americanus AAB00448, AAB00448, Aplysia californica matrilin-like 40 kDa protein Lehmannia valentiana ID with significant BLAST similarity (bold recovered from EMF and AG) Unknown Hemocyanin, Endoplasmin/Heat shock protein 90 kDa NP_001039228, Elongation factor 2 (EF-2) XP_001602460 similar to translation EF-2, Nasonia vitripennis LBP/BPI, LBP/BPI, gram-negative bacteria binding protein (GNBP) EF452345, Biomphalaria glabrata vWF domain containing sequence ATP synthase subunit, mitochondrial AAT06147, d representative coding sequences FC859944 ES748875 EX006505 CV548537 EE049678 ES484434 EX005363 FC859131 EE049679 EE722904 CF216480 DW474280 EV817465 ES745919 ES491466 c peptide sequences recovered TGYEDCIELKSTINYQGR, MNDIPCGTALK, VLCERDLFK, VIFQQALLNK LSSILLDR, SINAGIELK, SQGSSQIVR, QLAEELNR, RLHNEFSVIFLVFDK, LNSAFSPPSYSVWTTLLANVHDTLLEIAR NIQELTDGESNNLR, LLTVQFEQALSR, LGASWGIPQWDQTDESTALPK, LFSDPEDNPFYR, LQSETSADNFENIAGFHGAPNR GVVDSDDLPLNVSR, LGVIEDTSNRTR, FFSSNSDTEQTFLADYVER, EKQEAIYFIAGTSR, FQNIAKEGLNIDTSEK ICLKDLEEDHAGIPIK, VSEPVVSYR, ETVSAESEIMCLAKSPNK, FDFDVTEAR, EGVLCEENMR, GGGQIIPTAR, RVLYAAALTAEPR, GVVFDNQQIGNTPQITVKAHM VREWTEERLCR, VAVEADSDRR, LSAEMDEFKVDYSPVSK, VSVSQQSLEAR, HRGEVSWK, SDSTPSSQKPDDLPR, TFAESAHSHDYLK, VQLHGDVLVSTR, LSYVSSLIPELSSNSAGSVQVEVSSSK, LPKTNLR, VPDVEISEEGVR, FTHMVVR LLAEAHIEKNR, LIPDLLSYDVETSELTAIK, KGLPLMSLEDLSLLDPTVR ARIAEDTISEEDQK, FTHMIVR, FTHMVVREFAG, RGLPLMSLPDLR, EMDEFKVDYSPVSK, FAEDTISEEDQKEK, IAEDTISEQDQR, VSVSQQSLEAR IAEDTISEEDQKEK, LVPDILSYDVETR, SDSTPSSQKPDDLPR, TFAESAHSHDYLK, VPDVEISEEGVR, VQLHGDVLVSTR YGVVEVR, LMSNPTLR, DNVYGGWPR NEWLPTWR, GGFGGQNIWAGGER TGSWYDSFHTWR, GDETAMIVDYVEFR ISKGDWIWPAIWMLPR, KPWANNAPAPDVDFFNAR, GNEVGIGISQVSSTLHWG, PSPNENR AGIVLDDTLK, AAVSAGVNVLCAVXLR, TDQNAGLLAPVPFQCLGK, FGLVIFSDNAR, IFDLSTYNNHPELLEAILK, TPYLLGNTFTDK, ALVMANSLLTTTGRPGVPK, SPAHVFTTDEYK, NLQTLLYVTAQK, ECNGVVAPIEDDK, TLADIIFVLDSSGSIGADNYIK VVDLLAPYAR, IGLFGGAGVGK, TVLIMELINNVAK, AHGGYSVFAGVGER, EGNDLYHEMITTK, VSLVYGQMNEPPGARAR, VALSGLTVAEFFR, DQEGQDVLLFIDNIFR, b MW 15 150 100 100 60 55 55 50 50 a L,19) ( B) C) C) D) E) E) F) F) ( ( ( ( ( ( ( ( Sample B,M AG AG AG AG AG AG AG AG

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1. Hathaway et al. Page 23 , underlined NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Biomphalaria glabrata ) strains of M contain IgSF-like and conserved contain conserved C-type lectin-like domain (CTLD; contains IgSF-like and conserved ) strain or M line ( B C-type lectin-like (CTLD; NCBI CDD cd00037) domains ID with significant BLAST similarity (bold recovered from EMF and AG) C-type lectin C-type lectins Ribosomal protein S8 ABY87394 Haliotis diversicolor Unknown C-type lectins C-type lectin- like (CTLD; NCBI CDD cd00037) domains NCBI CDD cd00037) d that include the peptide sequence fragments listed. or egg mass fluid (EMF) of BB02 ( representative coding sequences FC859543 FC857569 ES747373 ES486406 FC857919, FC856131, FC859217 Biomphalaria glabrata Biomphalaria glabrata c ) from BB02 strain AG peptide sequences recovered FTQAGSEVSALLGR, GIAELGIYPAVDPLDSNSR, ILDPNVVGEEHYATAR MWGDYTTYLQEK, TQFIISDSFR, YYLNSR, TYFFTPNDAHFR KSLVIECR, YIEISSDFAGVVGR, DAVLTGSTSDTMSAFLK, TDEYKPQYDKCK, QGLVDLFFQK, TKSYNGR, ATGSLISGTDAEKER, TWLYHRSEK FELGRPPAHTKLGAK, QDHGNFSWGSEAITR, IDVVYNASNNEFVR, QWYEAHYATPLGR, GVKLSEEEEAVLNK, VEPALDEQFSTGR, ELEFYVR WYPYSQEYMK, IGLLSDQVMR, DTLLETFNFVR, MIINVDR, GKILLYVSTWDK, AGLALGQGAAFGNAAALAANLGLAK VIFQQALLNK, LYYASKDTVANFDK, SQLTWVFQR, MSVFDWIPGEPNDK, TGYEDCIELKSTINYQGR, MNDIPCGTALK, VLCERDLF, AGFLKNVNISIEIK, ILFQQALLNK, MNDISCNTALR LYYASKDTVANFDK, STTNYQGR VLCERDLFK, AGFLKNVNISIEIK b MW 34 35 33 25 25 18–15 a G) G,H) I) I) J, K) ( ( ( ( ( Sample AG AG AG AG AG Samples were prepared from either albumen gland ( in kDa. Genbank accession number are provided for relevant ESTs or coding sequences from combined result of MS and MS/MS, C.I. >0.95. a b c d letters or numbers in brackets refer to the gel sections indicated Fig. 2.

Dev Comp Immunol. Author manuscript; available in PMC 2011 April 1.