The effect of periodically "milking" to obtain Tyrian Purple from Plicopurpura pansa (Gould, 1853) on the frequency of expulsion and mortality.
KEY WORDS: purple snail, Plicopurpura pansa, Tyrian purple
The majority of purple-producing marine snails belong to the family of Muricidae, and most, if not all, produce in the hypobranchial gland (also called mucus gland) a colorless secretion, which is ejected into the mantle cavity, and turns on exposure to air and light to Tyrian purple (Fretter & Graham 1994). The secretion contains, besides a minute proportion of the precursors for the purple pigment, mucus, proteins, toxins, and narcotizing compounds. However, the hypobranchial gland of Plicopurpura pansa (Gould, 1853) is an exception among the muricids. It is so active that the snails can be stimulated periodically to expulse the secretion without being harmed (Rios-Jara et al. 1994; Michel-Morfin & Chavez, 2000).
The carnivorous, gonochoristic marine purple snail P. pansa inhabits intertidal rocky shores exposed to the high impact waves of the open sea. The range of distribution 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: average about 30 mm, but can reach a total shell length of 90 mm), and at low tides it is relatively easily gathered.
As in antiquity in the Old World, the use of muricids to obtain Tyrian purple for dyeing led also on the Pacific coast of Central America and Mexico to a product of high economic value. Here, mainly P. pansa is until now exploited by indigenous communities for Tyrian purple for dyeing cotton yarns and subsequently woven into traditional dresses (Turok 1999). The knowledge about the use of Tyrian purple from P. pansa before Columbus is limited. The first report known to me originates from Fernandez de Oviedo who visited in 1529 the peninsula of Nicoya (Costa Rica) and reported the use of "shells or oysters of purple" by natives to dye "mantas, cotton thread, and other things" (Fernandez de Oviedo [not dated], cited by Gerhard 1962). About thirty years later reported Vazquez de Coronado the use of shellfish dyed thread as a minor article of commerce in the province of Quepo in Costa Rica (Vazquez de Coronado 1563).
As Rios-Jara et al. (1994) and Michel-Morfin and Chavez (2000) reported can P. pansa be "milked" periodically to obtain Tyrian purple without harming the animal, however, too frequent milking caused mortalities. It is interesting however, to observe in reports that the exploitation of P. pansa for its pigment caused drastic declines of the snail population (Nuttall 1909, Turok & Acevedo 2000). Yes, in Nicoya, because of the increasing demands for purple dyed material the declining snail population led in 1760 to an uprising of the "indios" against the Spanish Alcalde (Fernandez-Guardia 1938, Jinesta 1940). In recent years with the increasing interest in 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 and the government permitted only Indian communities to follow the traditional exploitation of P. pansa for its pigment (Anonymous 1988, 1994).
But why did the populations of P. pansa decline so drastically? Is it because of too frequent "milking" without giving the snails time to recuperate? Is this causing mortalities or is it the stress from removing the snails from the rocks? Are there other reasons responsible? Low recovery rates of marked purple snails from areas of high wave actions point to the additional possibility that they are just washed away after milking by the waves (Ramirez-Rodriguez & Naegel 2003).
Information about the impact of periodically "milking" of the snails and survival, the percentage of animals showing secretion, and the time for recovery of the hypobranchial gland needed after being "milked" is a prerequisite for the assessment, planning, and management of the potential exploitation of P. pansa for its "Tyrian purple."
MATERIALS AND METHODS
About 300 adult snails were collected, during low tides between October and December 1999, from exposed low intertidal rocks at Playa Cerritos (23[degrees]19'54"N, 110[degrees]10'38"W) about 120 km southwest of La Paz, Baja California Sur, Mexico. The snails were transferred to the laboratory (CICIMAR, La Paz) and kept in inverted glass carboys with cut-off bottoms, each filled with 10 L of seawater (salinity: 30 [per thousand] to 34 [per thousand] daily water exchange; temperature range: 21[degrees]C to 23[degrees]C; 12 h light/dark). Sex determination was accomplished by visual inspection for a penis. During the experimental period the animals were fed to satiation with daily additions of squid.
For the here presented experiments (August 20 to November 25, 2003) 110 sexed and tagged animals were distributed randomly in 7 glass carboys. For the daily milking experiments one carboy was used with 8 males (length: 29.05 [+ or -] SD 4.31 mm) and 8 females (length: 30.97 [+ or -] SD 3.6 mm), one carboy with 15 makes (length: 26.8 [+ or -] SD 3.15 mm), and another with 15 females (length: 33.04 [+ or -] SD 6.62 mm); for the weekly, biweekly, three-weekly, and monthly milking experiments, the remaining four carboys contained 8 males and 8 females each. The size of all the 55 males ranged from 21.92-36.0 mm (27.9 [+ or -] SD 3.55) and of the females from 23.07-56.01 mm (32.7 [+ or -] SD 6.67). In the carboy with the 15 males for daily milking, on the 22nd day of culture one snail fell to the floor, slightly damaged its shell and died on the 27th day because of the damage. The same accident happened with one female snail, which dropped to the floor and damaged its shell after 20 days in culture and died after 39 days. It was recorded which marked animal expulsed secretion after slight mechanical pressure on the operculum. To determine the effect of daily removing the animals from the carboy without "milking" them, one carboy with 19 animals (8 males: 27.61 [+ or -] SD 2.78 and 9 females: 31.76 [+ or -] S.D. 5.35) was additionally installed.
To compare the proportion of animals that expulsed secretions from the hypobranchial gland under natural conditions, laboratory snails expulsion experiments were undertaken at Playa Cerritos in September 2003 (total number of snails collected 219), in October 2003 (total number of snails collected: 157), and in May 2004 (total number of snails: 455). In September and October 2003 additionally, the sex and total length of the animals were determined.
At the beginning of the experiments attempts were made to milk all 110 animals (55 males and 55 females). It was successful with only 30% (16 males and 17 females). No relation could be found between the size and sex of the animals and the frequency of secretions (Fig. 1).
[FIGURE 1 OMITTED]
From 23 male and 23 female snails, attempts were made to obtain daily secretions for a period of 98 days. During this period 1 male and 1 female died because of handling accidents and additionally 3 males and 2 females for unknown reasons.
At the beginning of the experiment (day 1), of the 46 animals, secretion was expulsed from only 9 (39.1%) males and 8 females (34.8%). The occurrence of secretions declined drastically during the experiment, and during the last weeks only few expulsions were noted (Fig. 2). No correlation could be noted between the sex of the snails and the decline of secretions
[FIGURE 2 OMITTED]
One interesting finding was, that the total number of secretions during the 98 days of the experiment was nearly double (122) from the surviving 20 females than that from the 19 males (68). During the test period each male excreted on average 3.6 times and each female 6 times. However, great individual differences could be observed by counting the total number of expulsions from each snail during the test period. During the 98 days five males did not expulse at all and one 11 times, one female 11 times, four females 6 times, and one not even once. No relation could be found between the size of the animals and the frequency of expulsion (Fig. 3). The smallest male with a shell length of 22.4 mm expulsed 6 times and the second largest one with a length of 32.48 mm 11 times. A similar picture can be seen with the female snails: the second smallest with a shell length of 24.62 mm expulsed 11 times and the second largest with a shell length of 40.16 mm 9 times. From these results it can be concluded that the frequency of expulsion is a characteristic of the individual snail and is not related to sex or size, although female snails expulsed more frequently than males.
[FIGURE 3 OMITTED]
To compare the proportion of expulsions of free-living animals with the laboratory snails, in September and October 2003, and in May 2004 experiments were undertaken at Playa Cerritos. In September 2003, 219 animals were collected; in October 157 animals; and in May 2004, 455 snails. Additionally in September and October 2003 the sex and total length of the collected animals were determined.
From the 831 animals collected 463 expulsed secretion (55.7%). From 376 sexed animals, 223 were males from which 123 expulsed (55.2%) and from the 153 females, 76 (49.7%) expulsed.
We tried to determine the effects of weekly, fortnightly, three-weekly, and monthly milkings on the frequency of secretion. At the beginning of the weekly experiment three expulsions were observed each from the eight male and eight female snails (31.4%). As in the experiment with the daily "milkings" the frequency of expulsions decreased drastically during the experiment. At the end of the experiment only one male and one female excreted (12.5%) (Fig. 4). The total number of secretions during the test period of 14 wk was significantly higher with the eight females (39) than with the eight males (27), but no relation between the size of the animals and the frequency of expulsion could be found. All animals survived the experimental period.
[FIGURE 4 OMITTED]
A similar situation could be found in the fortnightly "milking" experiment. At the start the experiment only two (25%) excretions were obtained from the eight males, and from the eight females only three (37.5%). The occurrence of expulsions declined during the test period. As an average, only 18% of the animals expulsed (Fig. 5). The number of the total expulsions during the test period was nearly double (13) by the female snails than by the males (7). Also in this experiment great individual differences were found, which were not related to the size, between the animals and the frequency of expulsions. From the eight males three animals didn't expulse during the test period, 4 animals once, and one animal three times. From the eight females four didn't expulse, two animals twice, one animal four times, and one animal even five times.
[FIGURE 5 OMITTED]
"Milking" every 3 wk and monthly had a different effect on the frequency of expulsions as reported before (Fig. 6). At the beginning of the three-weekly experiment with the eight females three expulsed, but with the eight males not one; however, at the end of the experiment the number of expulsions by males (12) and females (13) was nearly the same. Also here large differences could be observed between the individual frequency of expulsions. From the eight males one did not expulse at all during the experiment, and two expulsed three times. From the eight females two did not expulse at all, and one animal expulsed four times. As an average 31.5% of the animals expulsed during the test period and no decline in the frequency of expulsions could be found.
[FIGURE 6 OMITTED]
The effect of the period of recuperation between "milking" and the mortality: All animals in the weekly, fortnightly, three-weekly, and monthly experiment survived the "milking" during the experimental period of 98 days. However, a few mortalities were observed in the daily "milking" experiment. During handling of the snails on the 19th day of the experiment one female and one male dropped to the concrete floor and their shells were slightly damaged. The female died after 8 and the male after the l0 days. For unknown reasons another three males died on day 59, 62, and 70; and two females on day 69 and 76. The "milking" was not the reason for the mortalities. For the males the survival rate was 82.6%, for the females 87%.
To determine whether the daily removal of snails is causing mortalities, snails were removed daily from a carboy without "milking " them, and after a few minutes they were returned into the water again. During the test period of 98 days not one snail died because of the daily removal. This result shows that the removal of snails did not lead to mortalities.
One of the main functions of the hypobranchial gland is, according to Fretter and Graham, (1994) to produce secretion for trapping and cementing particulate matter sucked into the mantle cavity in the respiratory water current, prior to its expulsion. The gland is a thin band-like, inconspicuous glandular epithelium consisting of large (150-[micro]m) cells located at the superior part of the internal mantle cavity without excretory ducts, or organs to store the secretion. Like the salivary gland it secretes mucus continuously and can rapidly increase the secretion when stimulated. Together with the mucus minute amounts of the precursors of Tyrian purple are excreted, which lead on the surface of unwashed carboys to a purple coloration of the mucus (unpublished personal observations). When the animal contracts vigorously the cells of the hypobranchial gland burst open with their contents dispersed into the mantle cavity, where it mixes with the mucus and seawater (Lacaze-Duthiers 1859). Because of the few muscle fibers around the hypobranchial gland of muricids, the likelihood of mechanical stimuli is uncertain. The secretion also could be stimulated by neurosecretory activities because of the presence of neurosensory cells forming the hypobranchial nerve (Srilakshmi 1991), although there is no evidence available about a connection between the nerve and the secretory cells. In a histologic study, Bolognani-Fantin and Ottaviani (1981) observed that in the hypobranchial gland of M. brandaris there were six different cell types. Roller et al. (1995) studying by means of light and electron microscopy the hypobranchial gland of the estuarine snail Stramonita (= Thais) haemastoma canaliculata, described eight distinct cell types that are randomly distributed in the gland. Bolognani-Fantin and Otta Ottaviani (1981) questioned whether the great number of different cell types with different functions a characteristic of the hypobranchial gland of "Tyrian Purple," were producing muricids.
The volume of secretion from the hypobranchial gland released into the mantle cavity depends not only on the size but also on environmental and the physiologic conditions of the animals (Born 1936, Fouquet 1970). A large proportion of the volume of secretion obtainable from P. pansa after stimulation originates from the stored liquids of the mantle cavity. The concentration of the pigment precursors in the secretion of P. pansa could not, because of analytical difficulties, be quantitatively determined. Friedlander (1909) isolated 1.4 g of the pure pigment from 12,000 hypobranchial glands of Murex brandaris and showed that it was 6,6'dibromindigo. Using high performance liquid chromatography (HPLC) for the determination of the chemical composition of the final dye from P. pansa, it could be demonstrated that it consisted out of 90% 6,6'-dibromindigo, 9% monobromoindigo, and 1% dibromoindirubin (Withnall et al. 2003).
Repetitive stimulation to obtain the secretion from the hypobranchial gland without allowing a recuperation time of at least three weeks, results not only in a diminishing expulsed volume (Nuttall 1909, Michel-Morfin & Chavez 2000), but also, as demonstrated in this study, in an increase in the proportion of snails that didn't excrete at all. Michel-Morfin and Chavez (2000) reported an increase of snail mortalities with decreasing time of recuperation between the milkings. This is not consistent with the results presented here. The attempt to obtain daily secretions over a period of 98 days resulted in minor mortalities, mainly because of mistakes in handling the animals.
Differences between results concerning the proportion of expulsions obtained in the laboratory and the values obtained under natural conditions could be explained by the completely different environmental and physiologic conditions. The mucus production from the hypobranchial gland facilitates the locomotion of the animals. The snails in the field have to produce much more mucus because of the rough surface of the rocks than the animals cultured in the smooth glass carboys. Additionally, the snails in the laboratory were fed only squid, a food completely different compared with those from the intertidal zone, where the snails fed on other snails.
In mark and recapture experiments at Playa Cerritos the average percentage of first recapture was only 1%, probably because the marked snails deposited among the rocks where they were found did not adhere fast enough, and they were washed away to unsuitable areas by the high wave action. In contrast at a location with low impact waves, the average percentage of first recapture of marked snails was 22% (Ramirez-Rodriguez & Naegel 2003). The main reason for the declining snail P. pansa populations in the areas if "Tyrian Purple" production seems to be the likelihood of being washed away by high impact waves after "milking." For this reason the snails should never be removed from the rocks, and dyeing materials with Tyrian purple should only, in exceptional cases, be permitted to the indigenous people to allow the survival of an tradition.
The author expresses his thanks to C.A. Aguilar-Cruz (CICIMAR) for his technical assistance during the experiments, A. Rosales-M. and I. Castanon-E (U. Nayarit) for their help in determining the sex of the snails, and to J. Lopez-Rocha (CICIMAR), M. Delgado-T. (Tecnologico Culiacan) and O. Armenariz (CIBNOR) for improving the drawings. Special thanks to Chris Cooksey (London) for making suggestions to improve earlier versions of this manuscript and to the two anonymous reviewers for their comments. This work was supported by a grant from PIFI to C. A. Aguilar-Cruz and from COFAA and EDI to the author.
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LUDWIG C. A. NAEGEL *
Centro Interdisciplinario de Ciencias Marinas, Instituto Politecnico Nacional (CICIMAR/IPN) Apdo. Postal 592 La Paz, B.C.S. 23000 Mexico
* E-mail: firstname.lastname@example.org
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|Author:||Naegel, Ludwig C.A.|
|Publication:||Journal of Shellfish Research|
|Date:||Jan 1, 2005|
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