MICROSCOPY RESEARCH AND TECHNIQUE 00:000–000 (2012) Cytochemical Investigation of the Digestive Gland of Two Strombidae Species (Strombus gigas and Strombus pugilis) in Relation to the Nutrition JEAN-MARIE VOLLAND* AND OLIVIER GROS De´partement de Biologie, UMR 7138 SAE, Equipe Biologie de la mangrove, Universite´ des Antilles et de la Guyane, U.F.R des Sciences Exactes et Naturelles, B.P. 592. 97159 Pointe-a`-Pitre Cedex, Guadeloupe, France KEY WORDS molluscs; cytochemistry; physiology; lysosomal digestion; EFTEM ABSTRACT Strombus gigas and Strombus pugilis are threatened species and aquaculture rep- resents a good alternative solution to the fishing. In this study, we highlighted the intracellular digestion process in the digestive gland of two Strombidae species, S. gigas and Strombus pugilis, by the cytochemical characterization of two lysosomal enzymes: acid phosphatase and arylsulfa- tase. In order to check the efficiency of artificial food digestion, we conducted the characterization on freshly collected, starved and artificially fed individuals of S. pugilis. TEM observations of di- gestive gland sections from freshly collected individuals of both species revealed the presence of acid phosphatase and arylsulfatase activity mostly located in the apical third of digestive cells. Both enzymes were also detected in artificially fed individuals. In response to the starvation, acid phosphatase is not produced anymore by digestive cells, while arylsulfatase is still present. To our knowledge, this is the first cytochemical validation of intracellular digestion of artificial food in Strombidae. This study highlights the intracellular digestion of artificial food developed for Strom- bidae aquaculture. Moreover, we have shown that the lysosomal activity could be used as a feed index. Microsc. Res. Tech. 00:000–000, 2012. VC 2012 Wiley Periodicals, Inc. INTRODUCTION cations, the authors have studied the digestive gland Strombidae are marine benthic Gastropods which structure by histological observations and the quantifi- represent an important economic resource in the Ca- cation of the growing rate of individuals. However, to ribbean. At least four species represent a staple food our knowledge, no study reports an ultrastuctural including Strombus gigas LINNAEUS, 1758 and investigation of the digestive gland of Strombidae Strombus pugilis LINNAEUS, 1758. In 2001, for exam- except our previous works (Gros et al., 2009; Volland ple, 3,132 tones of S. gigas have been fished, which rep- et al., 2010, 2012). Strombidae are among the few Gas- resent more than 30 million USD (Aldana Aranda, tropods which present a crystalline style. Such struc- 2003). Due to an important fishing pressure, some spe- ture is more common in Bivalvia and is often associ- cies as S. gigas are threatened and some populations ated to a continuous microphagous nutrition (Fretter have already completely disappeared like in the Yuca- and Graham, 1962). The few studies which focused on tan (Mexico) for example. Natural stocks decrease rap- the digestion physiology of Strombidae reported the na- idly and international authorities have taken protec- ture of enzymes of the crystalline style (Alyakrinskaya, tion measures (Adams, 1970; Aldana Aranda, 2003; 2001; Horiuchi and Lane, 1965, 1966). To our knowl- Appeldoorn, 1987): S. gigas is included in the annex II edge, no cytochemical investigation of the digestive of the Convention on International Trade in Endan- gland of Strombidae has been conducted to date. Such gered Species (CITES) and also in the red list of the organ has a key role in the digestion process and repre- International Union for Conservation of Nature sents with the stomach the most complex part of the (IUCN). For these reasons, aquaculture represents a digestive tract (Owen, 1966). In this study, we high- good alternative solution to the high pressure on lighted the intracellular digestion process in the diges- Strombidae (Brito Manzano et al., 1998). While studies tive gland of two Strombidae species, S. gigas and report the development in laboratory of fertilized eggs Strombus pugilis, by the cytochemical characterization from the sea (Brito Manzano et al., 1999; Brito Man- *Correspondence to: Jean-Marie Volland, De´partement de Biologie, UMR 7138 zano and Aldana Aranda, 2004), to our knowledge no SAE. Equipe Biologie de la mangrove, Universite´ des Antilles et de la Guyane, U.F.R des Sciences Exactes et Naturelles, B.P. 592. 97159 Pointe-a`-Pitre Cedex, studies report a complete reproduction cycle of Strom- Guadeloupe, France. E-mail: [email protected] bidae in the laboratory. Veliger larvae are obtained and Received 3 March 2012; accepted in revised form 26 April 2012; accepted in grown up to the metamorphosis by feeding with unicel- revised form 26 April 2012 lular algae. Then juveniles are bred with artificial food Contract grant sponsor: ECOS-Nord (Caracte´risation reproductive, mole´cu- provided by small pellets. Few studies have investi- laire, e´cologique Apicomplexa-Strombidae, implication pour la peˆche et l’aqua- culture dans la re´gion Caraı¨be et le Golfe du Mexique); Contract grant number: gated the efficiency of unicellular algae and pellets for M09-A02 Strombidae breeding (Aldana Aranda and Sua´rez, DOI 10.1002/jemt.22074 1998; Aldana Aranda et al., 1997, 2007). In these publi- Published online in Wiley Online Library (wileyonlinelibrary.com). VC 2012 WILEY PERIODICALS, INC. 2 VOLLAND AND GROS of two lysosomal enzymes: acid phosphatase and aryl- Light and Transmission Electron Microscopy sulfatase. In order to check the efficiency of artificial Semithin sections (0.5 lm thick) were cut from the food digestion, we conducted the characterization on resin-embedded samples and stained with 0.5% tolui- freshly collected, starved and artificially fed individu- dine blue in 1% borax for light microscopy observation. als of Strombus pugilis. Ultrathin sections (60 nm) were obtained from resin blocks and observed without supplementary contrast MATERIAL AND METHODS with Energy Filtered Transmission Electron Micros- Experimental Conditions copy (EFTEM). Acquisitions in spectra mode (EELS, Individuals of S. gigas were collected during the Electron Energy Loss Spectroscopy) were performed authorized fishing period by professional fishermen on using a LEO 912 Omega transmission electron micro- or near Thalassia testudinum sea grass beds in Le Gos- scope (LEO Electron Optics GmbH, Oberkochen, Ger- ier, Guadeloupe (French West Indies). Strombus pugi- many) at 120 kV. Observation on image mode (ESI, lis samples were collected by hands on sand area in Electron Spectroscopic Imaging) was accomplished Saint-Franc¸ois, Guadeloupe. Living materials were with the ESIvision program (version 3.0 Soft-Imaging rapidly brought to the laboratory. Three S. gigas and Software, SIS, GmbH, 48153 Mu¨ nster). For the ele- three Strombus pugilis (batch Bcontrol) individuals were mental cartography, the subtractive method of the directly processed for cytochemical detection of lysoso- ‘‘three windows’’ was used (Jeanguillaume et al., 1978; mal enzymes. Six other Strombus pugilis were placed Reimer et al., 1992). in two 400 L raceway filled with sand-filtered sea water (pH 5 8; temperature 5 268C; psu 5 36; photoperiod 5 12 h) which was continuously oxygenated using an air Histochemical Detection of Lipids pump. Sea water was renewed twice a week and race- Digestive gland samples of S. gigas and Strombus way was cleaned to avoid development of microalgae pugilis were fixed for 6 h in 6% paraformaldehyde in and biofilm which could be used as food source. One sea water. Samples were then washed in sea water and batch named Bfed was fed ad libitum during 4 months transferred in 30% sucrose in sea water. They were with artificial food developed and provided by the CIN- then rapidly frozen in isopentane cooled at 2358C with VESTAV-IPN of Me´rida (Nutrition and Aquaculture of liquid nitrogen. Cryosections of 10 lm realized with a Molluscs Laboratories). It has been controlled visually Cryo-cut1 (American Optical Corporation) were that food pellets were ingested by animals. The other stained with black Soudan B as described by Gabe batch named Bstarved was kept 5 months in starvation. (1968). After their respective experimental maintenance, indi- viduals from Bfed and Bstarved were sacrificed and proc- essed for cytochemistry. RESULTS Ultrastructure of the Digestive Cells Characterization of Lysosomal Activity According to Gros et al. (2009), the digestive gland of Acid Phosphatase. Small digestive gland pieces Strombidae is composed of an assemblage of digestive were dissected and fixed for 2 h in 2% glutaraldehyde tubules and ducts. Digestive tubules are composed by and 1% paraformaldehyde in cacodylate buffer (0.1 M; three cell types: short pyramidal crypt cells, vacuolated 1,100 mOsm; pH 5 7.2). Pieces were then washed in cells, and the predominant long columnar digestive the same buffer and thick sections (50–100 lm) were cells (Figs. 1C and 2A). This last cell type is the most cut in refrigerated cacodylate buffer using an OCT important in terms of number and volume in the diges- Slicer1. The Gomori (1952) acid phosphatase detection tive tubules. They form a unistratified epithelium and method modified by Pasteels (1971) was used. Tissues are regularly aligned (Fig. 1A). Digestive cells were on slices were incubated in a 3.3 mg mL21 sodium ß-gly- average 90 lm long in S. gigas and 80 lm long in crophosphate solution saturated with lead nitrate. Strombus pugilis. They present a basal nucleus and Incubation was realized at 378C during 25 min. Slices three compartments can be distinguished in the cyto- were then rinsed in acetate buffer (sodium acetate 50 plasm: the basal third, the median third, and the apical mM, acetic acid 15 mM; pH 5 5) and in 3.5% acetic third (Figs. 1D, 2B, and 2C). The basal third is charac- acid before dehydratation through an ascending etha- terized by the presence of vesicles from 2 to 5 lm in di- nol series and embedded in a resin mixture composed ameter. Such vesicles appear empty on resin section, of Epon (63.2%) and Araldite (36.8%).