The hypobranchial gland from the purple snail Plicopurpura pansa (Gould, 1853) (prosobranchia: Muricidae).
KEY WORDS: hypobranchial gland, purple snail, Plicopurpura pansa, Muricidae
Most marine snails in the families Muricidae and Thaididae, which make up the genera Murex, Thais and Plicopurpura, produce in the hypobranchial gland (mucus gland) a viscous liquid secretion containing, besides mucus and biologically active compounds, minute amounts of chromogens. These develop enzymatically in light and oxygen into a purple pigment known as "Tyrian Purple," Royal Purple" or "Shellfish Purple." Fretter and Graham (1994) considered the main function of the hypobranchial gland to be a secretor of mucus for trapping and cementing particulate matter sucked into the mantle cavity with the respiratory water current prior to its expulsion.
The carnivorous, gonochoristic, marine, muricid purple snail Plicopurpura pansa (Gould, 1853) inhabits intertidal rocky shores exposed to high impact waves of the open sea. The range of P. pansa extends from the northwest coast of Mexico (Baja California Sur) (Clench 1947, Keen 1971) to northern Peru (Pena 1970, Paredes et al. 1999). The snail is not too small (shell length averages 30 mm but can be as large as 90 mm), and at low tides it is relatively easily gathered. An exceptional property of P. pansa, in comparison with that of other muricids, is that it ejects its dye-producing liquid in such large quantity, that there is no need to kill the animal to obtain the "Tyrian Purple." Furthermore, the hypobranchial gland is so active that the snails can be "milked" periodically without harming the animals (Rios-Jara et al. 1994, Naegel 2005). For these reasons it is not unexpected that P. pansa is exploited for "Tyrian Purple" production, probably since pre-Colombian times. In recent years however, with increasing public awareness of natural colors, the commercial exploitation of P. pansa for dyeing kimonos with "Tyrian Purple" had reached in Mexico such levels as to threaten the survival of the species. In 1988 P. pansa had to be declared by the Mexican government a species under special protection (Anonymous, 1988; 1994). Despite these exceptional properties of P. pansa, first as a source for "Tyrian Purple" and secondly about the state of the endangered snail populations, little is known about the principal life-history features, and many basic biological questions remain. Until now studies are lacking on the histology of the hypobranchial gland of P. pansa. The objectives of this study are to gain a better understanding of the biological function of this gland (1) by examining the general morphological features of the P. pansa hypobranchial gland using compound light microscopy; (2) determining by histochemical means the inner-cellular sites of tryptophan and (3) comparing the results with previously published reports about the histology of the hypobranchial gland from other Muricidae and Thaidae.
MATERIALS AND METHODS
More than 100 specimens of an unexploited population of P. pansa were collected in 2001 from intertidal rocks on days during extreme low-water spring tides at Playa Cerrito on the Pacific coast (23[degrees]19'54"N and 110[degrees]10'38"W), about 80 km south-west of La Paz. The average size of the males was 25.87 mm (range 14.86-43.5 mm), of the females 30.14 mm (range 13-73.9 mm).
Injecting a 10% neutral, buffered, formalin solution into the snails preserved the tissue of the animals. About 24 h later in the laboratory eight animals were selected and removed from the shell, subsequently dehydrated in an alcohol series, cleared with butylated hydroxanisole, and embedded in paraffin. Longitudinal and transversal sections (5 [micro]m) were prepared of the bypobranchial gland, and according to the methodology by Humason (1979), stained with Ferric Hematoxylin-Eosin or Mallory Trichromic stain. To prove the presence of tryptophan, as the origin of the precursors for "Tyrian Purple" (Verhecken 1989), the histochemical method for the demonstration of tryptophan (Davenport 1960) was applied.
The hypobranchial gland of P. pansa is an antero-posteriorly elongated organ located at the internal surface area of the mantle, that folds on its posterior side near the rectum, right of the ctenidia and anterior to the kidney (Fig. 1). On dead animals it is easily distinguishable by the purple color that develops after the removal of the shell. Parallel to the hypobranchial gland in the same position can be found a black pigmented structure, presumably the anal gland.
[FIGURE 1 OMITTED]
The secretory epithelium, which forms the hypobranchial gland consists of at least six different and very long (156.7 [micro]m) nonciliated cell types: (a) eosinophilic cells with an irregular cytoplasmic texture, (b) very abundant goblet mucus cells, (c) cells with strong acidophilic granules, (d) cells with light acidophilic granules, (e) a few cells at the rectal area with very fine basophilic granules and (f) empty cells.
Figure 2 shows a section of the hypobranchial gland: the basal membrane with the longitudinal muscle cells, eosinophilic and goblet mucus cells and cells with acidophilic granules. At the right side of the photograph is shown the mantle cavity with secreted mucus and acidophilic granules. At the left side is shown the external cubical epithelium, which is in contact with the shell.
[FIGURE 2 OMITTED]
It was impossible to distinguish clearly the different histological regions of the hypobranchial gland because the different cell types were uniformly distributed in the gland. The exception was the rectal area--the only region where a cell type was found with very fine basophilic granules and where the number of mucus cells and cells with acidophilic granules was reduced. Along the glandular epithelium in the basal tissue were found a few blood vessels and a thin layer of longitudinal smooth muscle fibers.
The number of acidophilic granular cells in the hypobranchial gland differed markedly between different individuals, probably, because of the different stages of secretion. In the mantle cavity there was always a large quantity of mucus (Fig. 3), and only occasionally acidophilic granular secretory products.
[FIGURE 3 OMITTED]
Adjacent to the secretory epithelium of the hypobranchial gland is located an acinous glandular-like structure, which could be the rectal or anal gland. The acini are composed of small (10 [micro]m) nonciliated cubical cells with a large quantity of fine and dark staining basophilic granules. No products of secretion were observed in the lumen of the acini, nor in the duct, which connects to the mantle cavity, the rectum, or the exterior of the snail (Fig. 4).
[FIGURE 4 OMITTED]
Only the two cell types with acidophilic granules in the hypobranchial gland showed a histochemically strong positive reaction for tryptophan, indicating that in these cells there are high concentrations of the precursors or chromogens for "Tyrian Purple."
In the Old World the production and use of "Tyrian purple" was forgotten with the fall of Byzantium (Constantinople) in 1453 A:D. Therefore, it was a big surprise to the scientific community when more than 200 y later, in 1685, William Cole was informed that at the coast of Ireland "Tyrian Purple" from marine snails was still used to mark fine linen (Cole, 1685). After many trials with different snail species at Minehead in England he found the means to obtain the precursors of "Tyrian Purple" from the muricid Nucella lapillus, and the procedure for dyeing materials with them to obtain the final pigment. He made the important observation, first, that as soon as the colorless fluid is exposed to air and light it becomes immediately yellow and greenish. Soon afterwards it turns into deep emerald green, blue, deep blue, and finally reaches the purple color; and, second, that for this stepwise chemical process light and air are needed. However, 30 y before the discovery by W. Cole about the use of "Tyrian Purple" in Ireland, the English priest Thomas Gage (1655) reported from the New World, Nicoya (Costa Rica), about the commercial exploitation of the secretions of the hypobranchial gland of P. pansa to dye garments for rich Spaniards.
Lacaze-Duthiers (1859) showed that the precursors for the pigments are not formed in the kidney or in a vein, as thought during his time, but in a band-Iike glandular epithelium located at the superior part of the internal mantle cavity. However, because of missing acini and excretory ducts he considered the glandular epithelium not to be a gland. His histological studies of the hypobranchial epithelium of muricids and thaids raised the interest of many scientists to describe in more detail the histology of the hypobranchial gland.
Letellier (1890) observed in the hypobranchial epithelium of Purpura lapillus that the purple producing cells in the middle part of the purple band are much larger, than the neighboring cells. During the same year Bernard (1890) found well-developed innervations in the base of the hypobranchial epithelium of P. lapillus, and described that the activity of neuro-epithelial cells has an impact on the production of mucus.
Erspamer (1946) showed by histochemical methods that in muricids the purple precursors and the enzyme "purpurasi" are localized in the median zone of the hypobranchial body and are kept separate, so that no reaction occurs. Hunt (1973) grouped the many different secretory cell types into two main categories: (a) acid mucin cells and (b) goblet cells. He did not include the purple producing cells. Additionally, as Bernard (1890), he found nerve cells on the base of the gland. Astonishingly, these nerve cells are not in contact with the secretory or purple producing but with ciliated cells (Hunt 1973).
Bolognani-Fantin and Ottaviani (1981) in a histochemical study observed in the hypobranchial gland of M. brandaris different cell types: (a) granulated cells with large, and fine granules, (b) with a homogenous cytoplasm, (c) ciliated cells having no impact on the secretion and (d) empty cells. The different granulated cells, which are only found in the middle area, could reflect different stages of purple production. In another histochemical study of the hypobranchial gland of the marine muricid Morula granulata Srilakshmi (1991) described a similar basic arrangement of cells, like described before by Bolognani-Fantin and Ottaviani (1981) and noted the presence of neuro-sensory cells, which form the hypobranchial nerve. Roller et al. (1995) studying by means of light and electron microscopy the hypobranchial gland of the estuarine snail Stramonita (=Thais) haemastoma canaliculata defined three anatomicaly and histologically different areas with eight distinct cell types, which are randomly distributed in the gland. Among these only one is ciliated, a cell type with acidophilic granules. Others are secretory cells that release large amounts of mucus into the mantle cavity.
We observed the same basic types of cells and the great similarity between the previously described hypobranchial glands of different muricids and thaids. According to Bolognani-Fantin and Ottaviani (1981) the great number of different cells with different functions is a characteristic of the hypobranchial gland of "Tyrian Purple" producing species. Bolognani-Fantin and Ottaviani (1981) mentioned that the hypobranchial gland of a freshwater (Viviparus viviparus) and of a terrestrial (Pomatias elegans) snail species show a much simpler glandular structure. Bolognani-Fantin and Ottaviani (1981) observed additionally that the cells with fine acidophilic granules react positively to phenolic and indolic substances, which are considered as "Tyrian Purple" precursors. This was confirmed by Srilakshmi (1991) who found strong reactions for tryptophan and tyrosine in the hypobranchial gland. Bolognani-Fantin and Ottaviani (1981) and our results show that only the cells with acidophilic granules react positively to tryptophan. Therefore the acidophilic granulated cells found by Roller et al. (1995) in the hypobranchial gland of the muricid S. haemastoma canaliculata could be the purple producing cells.
Lacaze-Duthiers (1859) observed that when the animal contracts vigorously the cells massively open by mechanical or osmotic pressure dispersing their contents into the mantle cavity. Because of the minimal quantity of muscle fibers around the hypobranchial gland of all the earlier mentioned muricids, the likelihood of muscular stimuli is uncertain. Release of the secretion also could be stimulated by neurosecretory activities, because of the presence of neurosensory cells that form the hypobranchial nerve (Srilakshmi 1991). However, there is no evidence of a connection between the nerve and the secretory cells.
Bolognani-Fantin and Ottaviani (1981) mentioned the presence of picrophilic granules on both lateral sites of the hypobranchial gland that reacted with indole, but they could not relate them to the production of mucus or purple. Both authors also mentioned that in the hypobranchial gland of Murex brandaris no structural or chemical differences could be observed during the different seasons of the year.
The large number of different cell types and many possible chemical activities in the hypobranchial gland are an indication that the gland has multiple biological functions. These are yet to be discovered.
The authors acknowledge thankfully the support for this study from CONACYT (Project 31566-N) and from CGPI, COFAA and EDI (Instituto Politecnico Nacional, Mexico). A part of this study formed the basis for C.A. Aguilar-Cruz of his Licenciatura in Marine Biology. We are thankful for all the comments, critics, and for the "tweeking" of our English by Chris Cooksey (London) and by Dr. Carriker (University of Delaware).
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LUDWIG C. A. NAEGEL * AND CARLOS AUGUSTO AGUILAR-CRUZ
Centro Interdisciplinario de Ciencias Marinas, Instituto Politecnico Nacional (CICIMAR/IPN) Apdo. Postal 592 LA PAZ, B.C.S. 23000 Mexico
* Corresponding author. E-mail: Inaegel@ipn.mx