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

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 proteins (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.

Introduction

The fertilized eggs of many animal species undergo development within egg masses deposited in the external environment, and the molecules used to protect the developing embryos 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], [8], [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], [12], [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 polysaccharide galactogen 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], [23], [24], [25]. Although the protein composition has not been systematically studied, Bai et al. [26], [27], [28] found phenoloxidase activity in both the AG and perivitelline fluid of B. glabrata, and associated the activity with SDS-PAGE protein 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 and perivitelline 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 enzyme (endo-1,4-β-mannanase 1 and 2) and a calcium-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.