The relationship between seaweed diet and purple ink production in Aplysia dactylomela rang, 1828 (gastropoda: opisthobranchia) from Northeastern Brazil.ABSTRACT Aplysia dactylomela is a large marine opisthobranch o·pis·tho·branch n. pl. o·pis·tho·branchs Any of various marine gastropod mollusks of the subclass Opisthobranchia, characterized by gills, a shell that is reduced or absent, and two pairs of tentacles. gastropod gastropod, member of the class Gastropoda, the largest and most successful class of mollusks (phylum Mollusca), containing over 35,000 living species and 15,000 fossil forms. , which inhabits shallow tropical shoreline regions, eats red and green algae, and releases a purple ink when disturbed. Many functions have been proposed for this secretion and although there is no consensus on this, some of its constituents are believed to be derived from the red algal algal pertaining to or caused by algae. algal infection is very rare but systemic and udder infections are recorded. See protothecosis. algal mastitis the algae Prototheca trispora and P. diet, it may provide the snail with a substantial survival advantage. A. californica only produces ink when it ingests red seaweeds. In some locations of the Northeastern Coast of Brazil, A. dactylomela is seen feeding only on green seaweeds, and yet it releases the ink. The aim of this work is to investigate this contradiction by studying the feeding habits of A. dactylomela and assessing the relationship between the algal diet and the purple ink. Feeding habits were investigated by field observation and by analysis of gut contents. Purple ink production was monitored by histologic analysis of the ink gland from sea hares kept in water tanks, fed with either red or green seaweeds. Composition and protein profile of the purple ink also were studied. Homologies between seaweed components and the purple ink were sought for by immunodiffusion immunodiffusion /im·mu·no·dif·fu·sion/ (-di-fu´zhun) any technique involving diffusion of antigen or antibody through a semisolid medium, usually agar or agarose gel, resulting in a precipitin reaction. techniques. Our findings are that the sea hare A. dactylomela, likewise other Aplysia species, needs to consume red seaweeds to be able to secrete the purple ink. The proteins of the ink seem to be synthesized by the sea hare itself and are not obtained directly from the diet, as is the case for the ink pigments. KEY WORDS: Aplysia dactylomela, purple ink, ink gland, seaweed diet INTRODUCTION Aplysia dactylomela is a large marine opisthobranch gastropod which inhabits shallow tropical shoreline regions, eats red and green algae, and lays large numbers of fertilized fer·til·ize v. fer·til·ized, fer·til·iz·ing, fer·til·iz·es v.tr. 1. To cause the fertilization of (an ovum, for example). 2. eggs in string-like gelatinous gelatinous /ge·lat·i·nous/ (je-lat´i-nus) like jelly or softened gelatin. ge·lat·i·nous adj. 1. Of, relating to, or containing gelatin. 2. Resembling gelatin; viscous. masses close to the sea surface. It is hermaphroditic her·maph·ro·dite n. 1. An animal or plant exhibiting hermaphroditism. 2. Something that is a combination of disparate or contradictory elements. and nocturnally active, but may be exposed to sunlight as it rests during the day (Carefoot 1987). Sea hares probably are best known because of the purple ink they release when disturbed. This ink is secreted from the ink gland located on the edge of the mantle shelf, and the ability to produce purple ink is reported to be associated with a red seaweed- containing diet (Coelho et al 1998). Of the 37 species of sea hare from the Aplysia genus, 30 can secrete purple ink (Nolen et al. 1995). Many functions have been proposed for this secretion, such as camouflage, alarm signal, pheromone pheromone Any chemical compound secreted by an organism in minute amounts to elicit a particular reaction from other organisms of the same species. Pheromones are widespread among insects and vertebrates (except birds) and are present in some fungi, slime molds, and algae. , aposematism Aposematism (from apo- away, and sematic warning), perhaps most commonly known in the context of warning colouration, describes a family of antipredator adaptations where a warning signal (use of color patterns by prey animals to signal their distastefulness to predators), bile excretion, predator deterrent, and cue of danger (Johnson & Willows 1999). Probably the ink has more than one role in the biology of sea hares. Although there is no consensus on the biologic function of the ink, it is known that some of its constituents are derived from red algae in the diet (Prince et al 1998), and it provides the snail with a substantial survival advantage. Recent work by Prince et al. (1998) on the ink glands of A. californica has improved our understanding of ink gland structure and processing and secretion of purple ink. In addition to studies of the ecologic and biologic aspects of chemical defense in sea hares, special attention also has been paid to the isolation and characterization of new bioactive substances from the purple ink. Thus, many bioactive substances have been isolated from the purple ink, including proteins with antibacterial activity in A. punctata (Nistratova et al. 1992), antitumor an·ti·tu·mor also an·ti·tu·mor·al adj. Counteracting or preventing the formation of malignant tumors; anticancer. Adj. 1. and citolytic activities in Dolabella auricularia, (Kisugi et al 1989, Yamazaki et al. 1989a) and citolytic and antibacterial activities in A. kurodai (Yamazaki et al. 1989b, Yamazaki et al. 1990). In Brazil, antibacterial and hemagglutinating activities were described for the purple ink of A. dactylomela (Melo et al. 1998; Melo et al. 2000). On the northeastern Coast of Brazil, there is an abundance of seaweed species that support the great biodiversity of this tropical region. On some beaches there is a predominance of green seaweeds (mainly Ulva fasciata), whilst on others, red seaweeds prevail. On the beaches dominated by green seaweed, A. dactyomela is seen feeding mainly on them and yet releases the purple ink when disturbed. This fact could be intriguing, considering that other Aplysia species need to consume red seaweeds to be able to produce the ink. To solve this apparent contradiction this study investigates the feeding habits of A. dactylomela and assesses the relationship between the algal diet and the purple ink. MATERIALS AND METHODS Determination of Feeding Habits Feeding habits of A. dactylomela were observed in 2 beaches of Ceara State, Northeast of Brazil, between August 2000 and July 2001. One beach was particularly rich in the green seaweed Ulva fasciata (beach 1, 38[degrees]38'48"W and 3[degrees]41'24"S); whereas, the other was densely rich in the red species, particularly Hypnea musciformis and Gracilaria spp. (beach 2,39[degrees]25'45"W and 3[degrees]22'18"S). Feeding habits of sea hare were examined monthly during daytime at low tide by observing the seaweed species consumed in the field and by dissecting under a stereomicroscope ster·e·o·mi·cro·scope n. A microscope equipped for stereoscopic viewing. ster  e·o·mi the gut contents of the sea hares and capturing the images with a
digital camera (Sony-Mavica, Japan).
Histologic Analysis of the Ink Gland Specimens of A. dactylomela were collected during low tide and transported to the laboratory in a container with seawater. Some specimens were de-inked by squeezing them gently for a few minutes outside the water and then kept in seawater tanks for 15 days with unialgal diets (U. fasciata or Gracilaria sp.) to observe whether there was purple ink production. At the end of this period, sea hares were anaesthetized adj. 1. rendered For light microscopy, ink gland samples were fixed with Bouin mixture (saturated picric acid picric acid (pĭk`rĭk) or 2,4,6-trinitrophenol (trī'nī'trōfē`nōl), C6H2(NO2)3 solution, 40% formaldehyde, glacial acetic acid glacial acetic acid n. Acetic acid that is at least 99.8 percent pure. (15:5:1 v/v) and embedded in paraffin. Tissue sections (5 [micro]m) were stained with hematoxylin hematoxylin /he·ma·tox·y·lin/ (he?mah-tok´si-lin) an acid coloring matter from the heartwood of Haematoxylon campechianum; used as a histologic stain and also as an indicator. and eosin eosin /eo·sin/ (e´o-sin) any of a class of rose-colored stains or dyes, all being bromine derivatives of fluorescein; eosin Y, the sodium salt of tetrabromofluorescein, is much used in histologic and laboratory procedures. (Junqueira & Junqueira, 1983) and with bromophenol blue for detection of proteins (Pearse 1960). Histologic sections were observed under a microscope, and photographs were taken. Chemical Composition of Purple Ink Purple ink was analyzed for contents of water, protein (total nitrogen), reduced carbohydrate, lipid, and ash. For the water determination, 1g of ink was dehydrated de·hy·drate v. de·hy·drat·ed, de·hy·drat·ing, de·hy·drates v.tr. 1. To remove water from; make anhydrous. 2. To preserve by removing water from (vegetables, for example). in an oven at 100[degrees]C to 110[degrees]C to constant weight. Total nitrogen was determined in samples of freeze-dried purple ink by micro-Kjeldahl digestion (Baethgen & Alley 1989). The content of reduced carbohydrate was determined according to Dubois et al. (1956). Lipid content was determined by n-hexane Soxleht extraction, and ash was quantified by heating 1g of ink in a muffle furnace at 620[degrees]C for 18 h. Amino Acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. Analysis Dry purple ink (1 mg) was hydrolyzed in 1 mL 6 M HC1 with 1% phenol phenol (fē`nōl), C6H5OH, a colorless, crystalline solid that melts at about 41°C;, boils at 182°C;, and is soluble in ethanol and ether and somewhat soluble in water. (w/v), in a sealed glass tube under [N.sub.2], at 110[degrees]C for 22 h. After hydrolysis hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds. , HCl and phenol were removed by evaporation, and the residue was analyzed in a Biochrom 20 (Pharmacia-LKB) amino acid analyzer. Electrophoretic Profile of Ink Proteins Sodium dodecyl sulphate polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis n. A technique for determining the molecular weight of proteins, in which proteins that have been coated in an anionic detergent undergo electrophoresis in a polyacrylamide gel. (SDS-PAGE SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ) was performed in the presence of beta-mercaptoethanol according to the method of Laemmli (1970). Proteins were stained with silver nitrate silver nitrate (nī`trāt), chemical compound, AgNO3, a colorless crystalline material that is very soluble in water. The most important compound of silver, it is used in the preparation of silver salts for photography, in chemical (Blum et al. 1987). Bovine serum albumin (66.0 kDa), egg albumin (45.0 kDa), porcine porcine /por·cine/ (por´sin) pertaining to swine. porcine pertaining to pig. See also hog (1), swine. porcine circovirus 1 a nonpathogenic virus. pepsin pepsin, enzyme produced in the mucosal lining of the stomach that acts to degrade protein. Pepsin is one of three principal protein-degrading, or proteolytic, enzymes in the digestive system, the other two being chymotrypsin and trypsin. (34.7 kDa), bovine [beta]-lactoglobulin (18.4 kDa), and egg lysozyme lysozyme: see immunity. Lysozyme An enyme that was first identified and named by Alexander Fleming, who recognized its bacteriolytic properties. (14.3 kDa) were used as standards (Sigma Co., USA). Seaweed Protein Extraction and Determination Proteins were extracted from fresh samples of the green seaweed U. fasciata and the red species H. musciformis and Gracilaria sp. The samples were ground with a mortar and pestle A mortar and pestle is a tool used to crush, grind, and mix substances. The pestle is a heavy stick whose end is used for pounding and grinding, and the mortar is a bowl. The substance is ground between the pestle and the mortar. with the following buffers at 50 mM: Glycine-HCl, pH 2.6; Tris HCl, pH 7.0; and sodium borate, pH 9.0. The extracts of U. fasciata and H. musciformis were prepared at the proportion of 1:3 (m/v) and that of Gracilaria sp. at 1:5 (m/v). The extracts were filtered through a nylon tissue and centrifuged at 15,000 g for 10 rain at 4[degrees]C. The supernatants (crude extracts) had the protein content determined according to Bradford (1976) with bovine serum albumin (BSA 1. BSA - Business Software Alliance. 2. BSA - Bidouilleurs Sans Argent. ) as the standard (purchased from Sigma Co., USA). Anti-Purple Ink Polyclonal Antibody Anti-purple ink polyclonal antibody was developed in a 3-month-old albino albino (ălbī`nō) [Port.,=white], animal or plant lacking normal pigmentation. The absence of pigment is observed in the body covering (skin, hair, and feathers) and in the iris of the eye. rabbit, which was immunized by intramuscular injection with 1 mg of purple ink, previously dialyzed di·a·lyze tr. & intr.v. di·a·lyzed, di·a·lyz·ing, di·a·lyz·es To subject to or undergo dialysis. [Back-formation from dialysis. and freeze-dried and then dissolved in 1 mL of sterile saline solution containing incomplete Freund adjuvant adjuvant /ad·ju·vant/ (aj?dbobr-vant) (a-joo´vant) 1. assisting or aiding. 2. a substance that aids another, such as an auxiliary remedy. 3. , 1:1 v/v (Sigma Co.). Booster injections were given without adjuvant subcutaneously on the 14th, 21st, 28th, and 35th days after the primary injection, Immune serum was obtained by blood sampling from the marginal ear vein on the 21st, 28th, and 35th days. Immunodiffusion Assay Immunologic relationships between protein extracts from sea weeds and the purple ink were assessed by the Ouchterlony double diffusion method (Hudson & Hay 1989). The assays were done in 1% agar plates prepared with 20 mM sodium phosphate buffer, pH 7.0, containing 150 mM NaCl and 0.02% sodium azide. After solidification of the agar, wells were made with a Pasteur pipette, and 30 [micro]L of each seaweed extract was applied into each well and tested against the anti-purple ink antibodies applied in the central well. After incubation at room temperature for 48 hours, the gels were washed with 150 mM NaCl, dried, and stained with Coomassie brilliant blue for visualization of the precipitation arcs. RESULTS Feeding Habits Sea hares from beach 1 were frequently surrounded by the green alga U. fasciata, and to a much less extent by red alga species (Gracilaria spp. and H. musciformis). In beach 2, however, the snails were surrounded by red algae, mainly H. musciformis, and some brown species. The analysis of gut contents from specimens collected showed that the snails ate mainly those alga species surrounding them. Those from beach 1 had gut contents comprised of approximately 70% U. fasciata (and traces of red species); whereas, those from beach 2 contained over 90% H. musciformis (photography not included). Histologic Analysis of the Ink Gland Histologic analysis showed that ink gland tissues from animals whose gut contained mainly green algae had most vesicles empty. The same was observed with the animals kept on the U. fasciata diet in the laboratory tank. By contrast, the ink glands of animals from beach 2, which consumed mainly red alga species, had most vesicles full (Fig. 1). [FIGURE 1 OMITTED] Chemical Composition of Purple Ink The purple ink is composed of approximately 99.5% water. Purple ink proximal chemical analysis, on dry basis, is described in Table 1, being comprised mainly of proteins (over 60%). Regardless of diet, the ink had the same basic composition of protein. carbohydrate, lipid, and ash. Amino Acid Composition The results of amino acid composition (Table 2) showed that the purple ink contains high levels of acidic amino acids, glutamic, and aspartic acid and very low content of sulfur amino acids. Electrophoretic Profile of Ink Proteins The purple ink of sea hares from beaches 1 and 2 showed similar electrophoretic profiles in terms of mobility and intensity of protein bands (Fig. 2). Two prominent bands were observed at 60 and 45 kDa, several minor bands span the apparent molecular mass range 20-30 kDa, whilst others were approximately 15 and few below 14 kDa. [FIGURE 2 OMITTED] Serological serological pertaining to or emanating from serology. serological test one involving examination of blood serum usually for antibody. Relationship Between the Purple Ink and Seaweed Extracts The immunodiffusion assay showed that the anti-purple ink antibodies reacted with the ink but did not recognize any seaweed component extracted either with acid, neutral or alkaline buffer (photography not included). This indicates, therefore, that no immunologic reactivity exists between components of algal diet and the purple ink. DISCUSSION This study describes the relationship between the seaweeds consumed by the sea hare A. dactylomela and its purple ink. Although it is has been reported that the secretion of purple ink by sea hares is associated with their consumption of red seaweeds (Prince et al. 1998), specimens that consumed mainly green seaweeds (beach 1) also were seen to release the purple ink. This contradiction was solved when the analysis of gut contents showed the presence of traces of red seaweeds, indicating that minor consumption of red species was enough to support the ink production. To prove that A. dactylomela, like A. californica (Prince et al. 1998; Coelho et al. 1998), only produces ink when it ingests red seaweeds; laboratory experiments were conducted with sea hares receiving the green seaweed U. fasciata, or the red Gracilaria sp., as the sole source of food. Our light microscopy studies of the ink gland of sea hares consuming only U. fasciata showed that most of the vesicles were devoid of purple ink; whereas, individuals that consumed red seaweeds had full vesicles in the ink gland. These observations confirm the necessity of red-seaweed consumption for A. dactylomela, and probably other sea hares of the same genus, to be able to secrete the purple ink. The chemical analysis of the purple ink showed that, regardless of the main seaweed species consumed by the sea hare, the content of protein in the ink was the same. This could indicate that either diet, rich in green or red seaweeds, must be able to supply the amino acids necessary for ink protein synthesis. Our analysis of amino acid composition showed that the purple ink is rich in acidic amino acids and has very low content of sulfur amino acids, reflecting the amino acid composition of both seaweeds (Ramos et al. 2000). Likewise, the electrophoretic profile of the ink proteins was always the same, regardless of the sample origin (beach 1 or 2), time of the year, or sea hare age. The finding that the anti-ink antiserum antiserum /an·ti·se·rum/ (an´ti-se?rum) a serum containing antibody(ies), obtained from an animal immunized either by injection of antigen or by infection with microorganisms containing antigen. did not recognize any protein of the seaweed extracts suggests that the sea hare itself synthesizes the ink proteins and that they are not obtained directly from the diet, as is the case for the ink pigments (Troxler et al. 1981 ; MacColl et al. 1990). Coelho et al. (1998) previously suggested that the pigment of A. californica purple ink is of algal origin, but the proteins are not. Thus, there seems to be no doubt about the origin of the ink substances (pigments and proteins) in A. dactylomela. Nevertheless, the roles in the purple ink have not yet been completely elucidated. Several biologic activities have been described in vitro for isolated proteins (Kisugi et al. 1989; Yamazaki et al. 1989a; Yamazaki et al. 1989b; Yamazaki et al. 1990; Nistratova et al. 1992; Melo et al. 1998; Melo et al. 2000) and pigments (Wessels et al. 2000) from sea hare ink, and all give support to a defense role. CONCLUSIONS The sea hare A. dactylomela, and possibly other Aplysia species, needs to consume red seaweeds to be able to secrete the purple ink. The pigment is from algal origin; whereas, the proteins are not. The protein content and composition seem to be always the same, regardless of the seaweed species consumed.
TABLE 1.
Chemical analysis of the purple ink from Aplysia dactylomela.
Components % Dry Basis
Total protein (N x 6.25) 64.87 [+ or -] 2.56
Reduced carbohydrate 9.07 [+ or -] 1.84
Lipid 2.20 [+ or -] 0.40
Ash 2.72 [+ or -] 0.20
Values are means [+ or -] standard deviation of at least
triplicate analyses.
TABLE 2.
Amino acid composition of purple ink of Aplysia dactylomela
Amino g of Amino Acid in
Acid Residue 100 g of Dry Matter
Ala 6.03
Arg 5.07
Asx 11.77
Cys 1.43
Glx 11.95
Gly 6.48
His 1.12
Ile 3.68
Leu 7.10
Lys 4.70
Met 1.55
Phe 4.55
Pro 8.98
Ser 5.78
Thr 7.54
Trp ND
Tyr 5.46
Val 7.59
ND = not determined.
ACKNOWLEDGMENTS The authors thank PET/CAPES for a BS fellowship to L.E.A. Bezerra, CNPq for grants received and to the students R.C.N. Amorim, J.E.A. Beserra-Jr, and M. Thadeo for their assistance during the field work. LITERATURE CITED Baethgen, W. E. & M. M. Alley. 1989. A manual colorimetric col·or·im·e·ter n. 1. Any of various instruments used to determine or specify colors, as by comparison with spectroscopic or visual standards. 2. procedure for measuring ammonium nitrogen in soil and plant Kjeldalh digest. Commun. Soil Sci. Plant Anal. 20:961-969. Blum, H., H. Bier bier n. 1. A stand on which a corpse or a coffin containing a corpse is placed before burial. 2. A coffin along with its stand: followed the bier to the cemetery. & H. J. Gross. 1987. Improved silver staining of plant-proteins, RNA RNA: see nucleic acid. 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Bull. 37:2179-2182. Yamazaki, M., S. Tansho, J. Kisugi, K. Muramoto & H. Kamiya. 1989b. Isolation and characterization of a novel cytolytic factor in purple ink of the sea hare Aplysia kurodai. Cancer Res. 49:3834-3838. Yamazaki. M., H. Ohye, J. Kisugi & H. Kamiya. 1990. Bacteriostatic bacteriostatic /bac·te·rio·stat·ic/ (bak-ter?e-o-stat´ik) inhibiting growth or multiplication of bacteria; an agent that so acts. and cytolytic activities of purple ink from the sea hare. Dev. Comp. Immunol. 14:379-383. LUIS ERNESTO ARRUDA BEZERRA, (1) ANA FONTENELE URANO CARVALHO, (1) LUCIANA AIRES AIRES advanced imagery requirements exploitation system (US DoD) AIRES Automated Integrated Regulatory Examination System AIRES Automated Information Retrieval and Expert System AIRES Aerolíneas de Integración Regional BARREIRA, (1) VANESSA LUCIA Lucia frustration causes her to murder husband. [Ital. Opera: Donizetti, Lucia di Lammermoor, Westerman, 126–127] See : Madness RODRIGUES NOGUEIRA, (1) JOSE ROBERTO FEITOSA SILVA, (1) ILKA il·ka also ilk adj. Scots Each; every. [Middle English ilk a, each one : ilk (variant of ech, each; see each) + a, one, a MARIA VASCONCELOS (2) AND VANIA MARIA MACIEL MELO (1), * (1) Department of Biology Federal University of Ceara Campus Pici Fortaleza, Ceara-Brazil 60 455-760 (2) Department of Biochemistry and Molecular Biology Federal University of Ceara Campus Pici--Fortaleza, Ceara Brazil * Corresponding author. E-mail: vmmmelo@ufc.br |
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