THE HYPOBRANCHIAL GLAND from the PURPLE SNAIL PLICOPURPURA PANSA (GOULD, 1853) (PROSOBRANCHIA: MURICIDAE) Author(S): LUDWIG C

THE HYPOBRANCHIAL GLAND FROM THE PURPLE SNAIL PLICOPURPURA PANSA (GOULD, 1853) (PROSOBRANCHIA: MURICIDAE) Author(s): LUDWIG C. A. NAEGEL and CARLOS AUGUSTO AGUILAR-CRUZ Source: Journal of Shellfish Research, 25(2):391-394. Published By: National Shellfisheries Association DOI: http://dx.doi.org/10.2983/0730-8000(2006)25[391:THGFTP]2.0.CO;2 URL: http://www.bioone.org/doi/full/10.2983/0730-8000%282006%2925%5B391%3ATHGFTP %5D2.0.CO%3B2

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Journal of Shellfish Research, Vol. 25, No. 2, 391–394, 2006.

THE HYPOBRANCHIAL GLAND FROM THE PURPLE SNAIL PLICOPURPURA PANSA (GOULD, 1853) (PROSOBRANCHIA: MURICIDAE)

LUDWIG C. A. NAEGEL* AND CARLOS AUGUSTO AGUILAR-CRUZ Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional (CICIMAR/IPN) Apdo. Postal 592 LA PAZ, B.C.S. 23000 Mexico

ABSTRACT Results are presented on the histology of the hypobranchial gland of the marine muricid Plicopurpura pansa (Gould 1853). The general structure and secretory features were investigated using light microscopy and histochemical methods for the determination of tryptophan/indol. The hypobranchial gland of P. pansa is an antero-posteriorly elongated organ located on 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. In dead animals it is easily distinguishable by the purple color that develops after removing the shell. Parallel to the hypobranchial gland, in the same position, can be found a black-pigmented structure, presumably the anal gland. The secretory epithelium, which forms the hypobranchial gland consists of at least six different and very long (156.7 ␮m) cell types. It was impossible to distinguish clearly the different histological regions of the hypobranchial gland, because the different cell types were uniformly distributed throughout the gland, with the exception of the rectal area. The number of acidophilic granular cells differed markedly between animals, probably because of different levels of secretion. In the mantle cavity was always found a large quantity of mucus and only occasionally acidophilic granulated secretory products. Only the two cell types with acidophilic granules in the hypobranchial gland showed histochemically strong positive reactions for tryptophan, indicating in these cells high concentrations of the precursors for “Tyrian Purple.”

KEY WORDS: hypobranchial gland, purple snail, Plicopurpura pansa, Muricidae

INTRODUCTION spite these exceptional properties of P. pansa, first as a source for “Tyrian Purple” and secondly about the state of the endangered Most marine snails in the families Muricidae and Thaididae, snail populations, little is known about the principal life-history which make up the genera Murex, Thais and Plicopurpura, pro- features, and many basic biological questions remain. Until now duce in the hypobranchial gland (mucus gland) a viscous liquid studies are lacking on the histology of the hypobranchial gland of secretion containing, besides mucus and biologically active com- P. pansa. The objectives of this study are to gain a better under- pounds, minute amounts of chromogens. These develop enzymati- standing of the biological function of this gland (1) by examining cally in light and oxygen into a purple pigment known as “Tyrian the general morphological features of the P. pansa hypobranchial Purple,” Royal Purple” or “Shellfish Purple.” Fretter and Graham gland using compound light microscopy; (2) determining by his- (1994) considered the main function of the hypobranchial gland to tochemical means the inner-cellular sites of tryptophan and (3) be a secretor of mucus for trapping and cementing particulate comparing the results with previously published reports about the matter sucked into the mantle cavity with the respiratory water histology of the hypobranchial gland from other Muricidae and current prior to its expulsion. Thaidae. The carnivorous, gonochoristic, marine, muricid purple snail Plicopurpura pansa (Gould, 1853) inhabits intertidal rocky shores MATERIALS AND METHODS exposed to high impact waves of the open sea. The range of P. More than 100 specimens of an unexploited population of P. pansa extends from the northwest coast of Mexico (Baja Califor- pansa were collected in 2001 from intertidal rocks on days during nia Sur) (Clench 1947, Keen 1971) to northern Peru (Peña 1970, extreme low-water spring tides at Playa Cerrito on the Pacific Paredes et al. 1999). The snail is not too small (shell length aver- coast (23°19Ј54ЉN and 110°10Ј38ЉW), about 80 km south-west of ages 30 mm but can be as large as 90 mm), and at low tides it is La Paz. The average size of the males was 25.87 mm (range relatively easily gathered. An exceptional property of P. pansa, in 14.86–43.5 mm), of the females 30.14 mm (range 13–73.9 mm). comparison with that of other muricids, is that it ejects its dye- Injecting a 10% neutral, buffered, formalin solution into the producing liquid in such large quantity, that there is no need to kill snails preserved the tissue of the animals. About 24 h later in the the animal to obtain the “Tyrian Purple.” Furthermore, the hypo- laboratory eight animals were selected and removed from the shell, branchial gland is so active that the snails can be “milked” peri- subsequently dehydrated in an alcohol series, cleared with butyl- odically without harming the animals (Rios-Jara et al. 1994, Nae- ated hydroxanisole, and embedded in paraffin. Longitudinal and gel 2005). For these reasons it is not unexpected that P. pansa is transversal sections (5 ␮m) were prepared of the hypobranchial exploited for “Tyrian Purple” production, probably since pre- gland, and according to the methodology by Humason (1979), Colombian times. In recent years however, with increasing public stained with Ferric Hematoxylin-Eosin or Mallory Trichromic awareness of natural colors, the commercial exploitation of P. stain. To prove the presence of tryptophan, as the origin of the pansa for dyeing kimonos with “Tyrian Purple” had reached in precursors for “Tyrian Purple” (Verhecken 1989), the histochem- Mexico such levels as to threaten the survival of the species. In ical method for the demonstration of tryptophan (Davenport 1960) 1988 P. pansa had to be declared by the Mexican government a was applied. species under special protection (Anonymous, 1988; 1994). De- RESULTS The hypobranchial gland of P. pansa is an antero-posteriorly *Corresponding author. E-mail: [email protected] elongated organ located at the internal surface area of the mantle,

391 392 NAEGEL AND AGUILAR-CRUZ 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 posi- tion can be found a black pigmented structure, presumably the anal gland. The secretory epithelium, which forms the hypobranchial gland consists of at least six different and very long (156.7 ␮m) noncili- ated 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. Microphotograph of the hypobranchial gland. bm, basal It was impossible to distinguish clearly the different histologi- membrane; ec, empty cell; eoc, eosinophilic granulated cell; apc, aci- cal regions of the hypobranchial gland because the different cell dophilic granules; lm, longitudinal muscle; mc, mucus cell. Bar = 20 µm. types were uniformly distributed in the gland. The exception was the rectal area —the only region where a cell type was found with branchial gland showed a histochemically strong positive reaction very fine basophilic granules and where the number of mucus cells for tryptophan, indicating that in these cells there are high con- and cells with acidophilic granules was reduced. Along the glan- centrations of the precursors or chromogens for “Tyrian Purple.” dular epithelium in the basal tissue were found a few blood vessels and a thin layer of longitudinal smooth muscle fibers. DISCUSSION The number of acidophilic granular cells in the hypobranchial gland differed markedly between different individuals, probably, In the Old World the production and use of “Tyrian purple” because of the different stages of secretion. In the mantle cavity was forgotten with the fall of Byzantium (Constantinople) in 1453 there was always a large quantity of mucus (Fig. 3), and only A:D. Therefore, it was a big surprise to the scientific community occasionally acidophilic granular secretory products. when more than 200 y later, in 1685, William Cole was informed Adjacent to the secretory epithelium of the hypobranchial gland that at the coast of Ireland “Tyrian Purple” from marine snails was is located an acinous glandular-like structure, which could be the still used to mark fine linen (Cole, 1685). After many trials with rectal or anal gland. The acini are composed of small (10 ␮m) different snail species at Minehead in England he found the means nonciliated cubical cells with a large quantity of fine and dark to obtain the precursors of “Tyrian Purple” from the muricid Nu- staining basophilic granules. No products of secretion were ob- cella lapillus, and the procedure for dyeing materials with them to served in the lumen of the acini, nor in the duct, which connects to obtain the final pigment. He made the important observation, first, the mantle cavity, the rectum, or the exterior of the snail (Fig. 4). Only the two cell types with acidophilic granules in the hypo-

Figure 3. Microphotograph of the hypobranchial gland showing the Figure 1. Microphotograph of a section (5 µm) of the body of P. pansa. large quantity of secretory products in the mantle cavity. ec, empty bms, bucal mass; ct, ctenidium; dg, digestive gland; f, foot; hg, hypo- cell; eoc, eosinophilic granulated cell; apc, acidophilic granules; k, branchial gland. Bar =1 mm. kidney; mc, mucus cell; sp, secretory products. Bar = 100 µm. HYPOBRANCHIAL GLAND 393

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 es- tuarine snail Stramonita (=Thais) haemastoma canaliculata de- fined 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 acido- philic 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 simi- larity between the previously described hypobranchial glands of Figure 4. Microphotograph showing the hypobranchial gland, anal gland and rectum. ag, anal gland; hg, hypobranchial gland; k, kidney; different muricids and thaids. According to Bolognani-Fantin and sp, secretory products; r, rectum. Bar = 100 µm. 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) that as soon as the colorless fluid is exposed to air and light it mentioned that the hypobranchial gland of a freshwater (Viviparus becomes immediately yellow and greenish. Soon afterwards it viviparus) and of a terrestrial (Pomatias elegans) snail species turns into deep emerald green, blue, deep blue, and finally reaches show a much simpler glandular structure. Bolognani-Fantin and the purple color; and, second, that for this stepwise chemical pro- Ottaviani (1981) observed additionally that the cells with fine aci- cess light and air are needed. However, 30 y before the discovery dophilic granules react positively to phenolic and indolic sub- by W. Cole about the use of “Tyrian Purple” in Ireland, the English stances, which are considered as “Tyrian Purple” precursors. This priest Thomas Gage (1655) reported from the New World, Nicoya was confirmed by Srilakshmi (1991) who found strong reactions (Costa Rica), about the commercial exploitation of the secretions for tryptophan and tyrosine in the hypobranchial gland. Bolognani- of the hypobranchial gland of P. pansa to dye garments for rich Fantin and Ottaviani (1981) and our results show that only the cells Spaniards. with acidophilic granules react positively to tryptophan. Therefore Lacaze-Duthiers (1859) showed that the precursors for the pig- the acidophilic granulated cells found by Roller et al. (1995) in the ments are not formed in the kidney or in a vein, as thought during hypobranchial gland of the muricid S. haemastoma canaliculata his time, but in a band-Iike glandular epithelium located at the could be the purple producing cells. superior part of the internal mantle cavity. However, because of Lacaze-Duthiers (1859) observed that when the animal con- missing acini and excretory ducts he considered the glandular epi- tracts vigorously the cells massively open by mechanical or os- thelium not to be a gland. His histological studies of the hypo- motic pressure dispersing their contents into the mantle cavity. branchial epithelium of muricids and thaids raised the interest of Because of the minimal quantity of muscle fibers around the hy- many scientists to describe in more detail the histology of the pobranchial gland of all the earlier mentioned muricids, the like- hypobranchial gland. lihood of muscular stimuli is uncertain. Release of the secretion Letellier (1890) observed in the hypobranchial epithelium of also could be stimulated by neurosecretory activities, because of Purpura lapillus that the purple producing cells in the middle part the presence of neurosensory cells that form the hypobranchial of the purple band are much larger, than the neighboring cells. nerve (Srilakshmi 1991). However, there is no evidence of a con- During the same year Bernard (1890) found well-developed inner- nection between the nerve and the secretory cells. vations in the base of the hypobranchial epithelium of P. lapillus, Bolognani-Fantin and Ottaviani (1981) mentioned the presence and described that the activity of neuro-epithelial cells has an of picrophilic granules on both lateral sites of the hypobranchial impact on the production of mucus. gland that reacted with indole, but they could not relate them to the Erspamer (1946) showed by histochemical methods that in mu- production of mucus or purple. Both authors also mentioned that in ricids the purple precursors and the enzyme “purpurasi” are local- the hypobranchial gland of Murex brandaris no structural or ized in the median zone of the hypobranchial body and are kept chemical differences could be observed during the different sea- separate, so that no reaction occurs. Hunt (1973) grouped the many sons of the year. different secretory cell types into two main categories: (a) acid The large number of different cell types and many possible mucin cells and (b) goblet cells. He did not include the purple chemical activities in the hypobranchial gland are an indication producing cells. Additionally, as Bernard (1890), he found nerve that the gland has multiple biological functions. These are yet to be cells on the base of the gland. Astonishingly, these nerve cells are discovered. not in contact with the secretory or purple producing but with ciliated cells (Hunt 1973). ACKNOWLEDGMENTS Bolognani-Fantin and Ottaviani (1981) in a histochemical study observed in the hypobranchial gland of M. brandaris differ- The authors acknowledge thankfully the support for this study ent cell types: (a) granulated cells with large, and fine granules, (b) from CONACYT (Project 31566-N) and from CGPI, COFAA and with a homogenous cytoplasm, (c) ciliated cells having no impact EDI (Instituto Politécnico Nacional, México). A part of this study 394 NAEGEL AND AGUILAR-CRUZ formed the basis for C.A. Aguilar-Cruz of his Licenciatura in for the “tweeking” of our English by Chris Cooksey (London) and Marine Biology. We are thankful for all the comments, critics, and by Dr. Carriker (University of Delaware).

LITERATURE CITED Anonymous. 1988. Acuerdo intersecretarial que regula el desarrollo, con- Hunt, S. 1973. Fine structure of the secretory epithelium in the hypobran- servación y aprovechamiento de la especie denominada caracól Pur- chial gland of the prosobranch gastropod mollusk Buccinum undatum pura pansa, en beneficio de los núcleos de población que tradicional- L. J. mar. Biol. Ass. U.K. 53:59–71. mente lo han explotado y dispone las medidas necesarias para la preser- Keen, A. M. 1971. Sea shells of tropical West America: Marine molluscs vación de las costumbres y tradiciones derivadas del aprovechamiento from Baja California to Peru. Stanford, CA: Stanford University Press. del molusco. In: Diario Oficial de la Federación, Secretaría de Pesca, 1064 pp. March 30,1988. México, D.F. pp. 10–12. Lacaze-Duthiers, H. 1859. Memoire sur la pourpre. Annales de Sciences Anonymous. 1994. Norma Oficial Mexicana NOM-ECOL-059-1994, que Naturelles 4 ser. Zoologie 12:5–84. determina la especies y subespecies de flora y fauna silvestres y acuáti- Letellier, A. 1890. Recherches sur la pourpre produite par le Purpura cas en peligro de extinción, amenazadas, raras y las sujetas a protección lapillus. Archives de Zoologie experimentale et generale No. 3, 2 ser. especial y que establece especificaciones para su protección. In: Diario Vol. 8. 361–407. Oficial de la Federación. May 16, 1994. México, D.F. pp. 2–56. Naegel, L. C. A. 2005. The effect of periodically “milking” to obtain Bernard, F. 1890. Glande a mucus. Sécrétion du mucus. Chapter 1: His- Tyrian Purple from Plicopurpura pansa (Gould, 1853) on the fre- tologie de glande a mucus. In: Recherches sur les organs palléaux. quency of expulsion and mortality. J. Shellfish Res. 24(1):85–90. Annales de Sciences Naturelles 7 ser., Zoologie, Vol 91. pp. 296–305. Paredes, C., P. Huaman, F. Cordoso, R. Vivar & V. Vera. 1999. Estado Bolognani-Fantin, A.M. & Ottaviani, E. 1981. The hypobranchial gland of actual del conocimiento de los moluscos acuáticos en el Perú. Rev. some prosobranchia (Mollusca, Gastropoda) living in different habi- Peruana Biol. 6(1):5–47. tats: a comparative histochemical study. Monitore zool. Ital. (NS) 15: Peña, G. G. M. 1970. Zonas de distribución de los gasterópodos marinos 63–76. del Perú. Anales Científicos de la Universidad Nacional Agraria 8: Clench, W. J. 1947. The genera Purpura and Thais in the western Atlantic. 153–170. Johnsonia 2(23):61–91. Cole, W. 1685. A letter from Mr. William Cole of Bristol, to the Phil. Rios-Jara, E., H. G. León-Alvarez, L. Lizárraga-Chávez & J. E. Michel- Society of Oxford; containing his observations on the purple fish. Phi- Morfín. 1994. Producción y tiempo de recuperación del tinte de Pli- los. Trans. Roy. Soc. London 15(178), 1278–1288. copurpura patula pansa (Neogastropoda: Muricidae) en Jalisco, Davenport, H. A. 1960. Histological and histochemical technics. Philadel- México. Rev. Biol. Trop. 42(3):537–545. phia, PA: W. B. Saunders Company. 401 pp. Roller, R. A., J. O. Rickett & W. B. Stickle. 1995. The hypobranchial gland Fretter, V. & A. Graham. 1994. British Prosobranch Molluscs. Their func- of the estuarine snail Stramonita haemastoma canaliculata (Gray) tional anatomy and ecology. Revised and updated edition. London: The (Prosobranchia: Muricidae): a light and electron microscopical study. Ray Society. 820 pp. American Malacological Bulletin 11(2):177–190. Gage, T. 1655. A new survey of the West-Indias: or, The English American Srilakshmi, G. 1991. The hypobranchial gland in Morula granulata (Gas- his travail by sea and land. 2nd ed. London: E. Coses. pp. 192–193. tropoda: Prosobranchia). J. mar. bioi. Ass. U.K. 71:623–634. Humason, G. L. 1979. Animal tissue techniques. W.H. Freeman and Comp. Verhecken, A. 1989. The indole pigments of mollusca. Annls. Soc. R. Zool. San Francisco, CA. 560 pp. Belg. 119(2):181–197.