Reproductive biology of the "Copey" snail Melongena melongena (Linnaeus, 1758) in Cispata Bay on the Caribbean coast of Colombia.

ABSTRACT The reproductive cycle of the "Copey" snail Melongena melongena at Cispata Bay was examined from October 1998 to September 1999. Gonad maturation was studied through macroscopic and microscopic observations on the gonads of females >70 mm (total shell height), showing that individuals were mature throughout the year, but with a period of maximal maturity in March which coincided with the time of maximum recruitment. The mean size at first maturity was 52 mm for males and 65 mm for females. A small number of intersex individuals (4%) (masculinized females, with a normally developed ovary and a rudimentary penis; feminized males with a normally developed testis and gonopore) occurred at intermediate sizes. The proportion of males in the population decreases with the increase in individual size, with only females present at the biggest sampled size-class (mean size of 98.5 mm shell height), suggesting protandric hermaphroditism. If this is proven to be true, artisanal fishing preferring larger animals, could severely interfere in the success of reproduction.

KEYWORDS: Reproductive cycle, Melongena melongena, Caribbean, protandric hermaphroditism, intersex,.

INTRODUCTION

The "Copey" snail Melongena melongena (Linnaeus, 1758) is widely distributed in the Gulf of Mexico, the Caribbean and the Antilles. The snail lives on the shallow areas of coastal lagoons, mangroves and estuaries, being an important component of the fauna on soft or muddy bottoms. The snail tends to concentrate on areas with high densities of its prey, which are bivalves, other gastropods, ascidians and carrion (Hathaway & Woodburn 1961, Hawkins 1973, Rodriguez 1976, Flores 1980, Cosel 1986, Morton 1986, Dalby 1989, Villareal 1989, Bowling 1994). Considering this behavior, fishers sometimes use mangrove roots, covered with the mitilid Mytilopsis soller as bait, letting the snails aggregate on the bait from one day to the next. The method of collection of these snails includes walking over the bottom and locating the snails beneath the mud with the foot. By doing this, the fishers are not able to discriminate size, all sizes being fished. M. melongena attains sizes up to 200 mm of shell height (Clench & Turner 1956, Abbott 1974, Flores 1980, Diaz & Puyana, 1994). In Cispata Bay individuals between 12 and 151 mm shell height are found, the mean size of capture being 57 mm.

M. melongena copulates from December to July in Colombia (Rodriguez 1976) and then produces egg capsules. Egg masses of M. melongena have a common basal layer. The number of capsules produced fluctuates between 27-31, located along a string which measures up to 218 mm in length. Some modified, sterile egg capsules are produced and serve to anchor the egg mass on the soft bottom. This sterile capsules are smaller than fertile capsules, but have the same shape and are spaced approximately 28 mm apart at one end of the string. The average distance between fertile capsules along the string is only 6 mm, this distance getting shorter towards the last capsules produced. Egg masses are mostly buried in soft bottoms close to the vegetation on the coast. The capsules contain al least 185 to 290 embryos according to D'Asaro (1997), or 300 to 400 embryos according to Flores (1980), each embryo measuring 250 to 300 [micro]m. The development within the capsules takes 20 to 25 days. By day 16 a well developed veliger larvae is observed, having four large velar lobes with a double row of strong cilia on their outer edges. The foot and operculum are well developed, the larvae apparently being able to crawl. By day 25 or older, when removed from the capsule, the larvae no longer protruded the velar lobes, but they crawl actively and could rapidly turn over the shell with the aid of the foot. When the capsules open, most of the young snails, now incapable of swimming, immediately crawl and appeared to be feeding (Clench and Turner 1956)

Rodriguez (1976) reported that the smallest mature females found in Cartagena Bay measured 80 mm and the males 65 mm of shell height. Besides this observations by Rodriguez, information on the gonad maturation of M. melongena in Colombia is scarce.

With few exceptions, prosobranch gastropods are gonochoric, typically with internal fertilization, although some species demonstrate protandric hermaphroditism (Gallardo 1989). Rodriguez (1976) identified M. melongena females with shell heights between 40 and 62 mm having an incipient development of the penis. Species related to M. melongena, such as Busycon carica and M. corona, have similar characteristics (Castagna & Kraeuter 1994, Zetina 1999).

Previous observations have suggested that in nursery habitats, small individuals were mostly males and in areas where growth and maturation occur, larger individuals were predominantly females (Hathaway & Woodburn 1961, Woodbury 1986, Zetina 1999). Intersex individuals and sex dominance in different size categories, suggest that M. melongena may be a consecutive protandric hermaphrodite. This requires verification, since artisanal fishing activities, which prefer larger sizes, may negatively affect the population by seriously altering sex proportions. The fishery for M. melongena occurs throughout the Caribbean, but as the snail is only used for local consumption, it does not appear in the statistics. Nevertheless, the fishery is important, for example Hernandez (2001) estimating for the Cispata Bay area (the bay and nearby sloughs) that 70% or the total biomass of the snail population is fished, representing for local fishers ca. 30% of their total income. The meat of the snail is mainly used around the Caribbean to offer appetizers (the popular "botanas") in beach restaurants of tourist areas.

The objective of the present study was to describe the reproductive cycle and pattern of recruitment, sex ratio and the presence of intersex individuals in M. melongena in Cispata Bay. This in formation should provide a base for fisheries resource administrators to design biologically sound management measures for this resource.

MATERIALS AND METHODS

Study Area

Individuals of M. melongena were collected from October 1998 to September 1999 from the Cispata Bay area (includes the nearby sloughs and the area close to Amaya Beach) (Fig. 1).

[FIGURE 1 OMITTED]

Reproductive Cycle

Each month from October 1998 to September 1999, snails were searched for in the field (as fishers do, with the foot), collected manually and measured. This sampling was part of a study regarding distribution, abundance and growth of the snail. Thus, a great number of individuals (over 100), representing the entire depth distribution, was sampled. For the study of the reproductive cycle the largest 30 females, but measuring at least 70 mm of shell height (females of that size were observed to lay eggs), were selected. Due to the fishery, in which the average size of capture is around 50 mm, with few individuals reaching 80 mm or more, large individuals are scarce in the field, so when ever possible, larger individuals were bought from fishers. To get the 30 females, collected individuals had to be sacrificed to verify sex. As the aim of the study was to know the time of reproduction of M. melongena in Cispata Bay, the analysis was concentrated on females, considering that only when they are mature, copulation can occur. As perhaps not all females, but probably all males, may reproduce each year (as described for other gastropods), this method may sub estimate the number of reproducing individuals within the population. But the general seasonal pattern will not be affected by this.

The tissues of each measured individual (total shell height in mm), were removed, and the gonad separated from them. Each component was then weighed separately (drained wet) to the nearest 0.01 g. The gonads were fixed in Bouin's fluid and prepared using traditional histologic procedures to obtain thin sections (6 [micro]m) stained with Ehrlich's Haematoxylin and eosin. Three sections were taken from each embedding block, each 360 [micro]m apart to avoid including the same reproductive follicles in the different sections.

Two methods were used to establish the reproductive cycle. The first included a macroscopic quantitative evaluation (gonad index = GI) and the second was based on microscopic qualitative evaluation (index of gonad maturity = IGM).

The gonad index (GI) is represented by:

GI = (weight of the gonad / total weight of tissues) x 100

For the total weight of tissues all soft tissues of the animal, including head, foot with operculum, visceral mass, mantle, as well as the gonad, were included.

The index of gonad maturity (IGM) characteristics were established for each stage of gonad development, as defined by Ramorino (1975):

* Evacuated (I): walls of acini with a corrugated appearance and abundant phagocytes within them.

* Maturing (II): internal walls of the acini with pedunculated and pyriform vitellogenous oocytes and the presence of a few phagocytes and large spaces between them.

* Maximum maturity (III): the walls of the acini take on a polygonal form without spaces between them and the presence of abundant nutritive cells.

IGM was calculated using the following equation:

IGM = ([SIGMA] (n*F)/N) x 100

Where F = stage of gametic development (I-III), n = number of individuals in stage F and N = number of total individuals.

Recruitment

Using the size frequencies obtained throughout one sampling year and the growth parameters calculated by Hernandez (2001), the FISAT program routine for recruitment patterns (Gayanilo & Pauly 1997) was used. This permits determination of the number of recruitment pulses per year and their intensities. For this study, recruitment was defined as the addition of new individuals into the benthic population.

Mean size at first maturity

Based on information reported by Rodriguez (1976) and Flores (1980), which describe that M. melongena copulates from December to June, the egg masses being most abundant in February and March, an intensive sampling was done between December 1998 and March 1999. Ten individuals were obtained for each 10 mm size class, beginning with 20 mm individuals. This number of individuals was decided, considering the possibility to really obtaining them in the field, or being able to buy them from fishers. The lower limit of 20 mm corresponds to the smallest individuals fishers include in their catch. Both males and females were identified, using histologic sections of the gonads. Individuals were rated as either immature (abundant connective tissue; absence of, or very few acini), or mature (Stages I-III). The data for each sex were fitted to a logistic model using the CurveExpert 1.3 program (Copyright (c) 1995-1997 Daniel Hyams.) based on the equation:

Y = a/(1 + [be.sup.-cx])

Where Y = Relative accumulated frequency (%), a = constant, b = slope, c = constant and x = total shell height.

The mean size at first maturity was estimated at a level of 50% of the relative accumulated frequency (Zetina 1999).

Sex ratio and Intersex individuals

Through macroscopic and microscopic observations, individuals were identified as either males or females and thereby an estimate of the sex ratio was obtained. When sex determination between macroscopic and microscopic examination did not agree, individuals were classified as in the intersex phase based on criteria used by Reed (1993) and Zetina (1999). At the macroscopic level, masculinized females present an atrophied penis which was smaller than that of normal males. An ovary was observed at the microscopic level. The feminized male had a gonopore with no macroscopic differences from that of a normal female, and microscopically they have a testis.

RESULTS

Cispata Bay (Fig. 1) is located in the Department of Cordoba, on the Caribbean coast of Colombia, between 90[degrees]26' and 9[degrees]21'N and 57[degrees]54' and 75[degrees]45'W. The bay is located south of the Gulf of Morrosquillo. It has an irregular topography, is bordered by mangroves, and has estuarine waters with a mean depth of about 2 m. (Sanchez et al. 1997). Inland from the bay occur 12 sloughs which connect with it by channels (Fig. 1).

[FIGURE 1 OMITTED]

The study area experiences four climatic seasons including, (a) the dry season from January through March, (b) transition to the rainy season from April through August, (c) the rainy season from September through November and (d) transition to the dry season in December. These seasons are affected by the Caribbean Current which flows from east to west and by the Darien Countercurrent, or Panama Current, which flows northward along the coast (Ramirez 1994). The tides are semidiurnal, with an amplitude of less than 1 m; the annual mean temperature is 26.7[degrees]C and the annual precipitation is between 900 and 1200 mm (Patino & Flores 1993).

Reproductive cycle

A high proportion (>50%) of mature individuals (Stage III) were observed from January through September, except for the months of February, April, and June in which the proportion diminished (Fig. 2). Each peak of mature individuals was preceded and followed by maturing individuals (Stage II). Spawned individuals (Stage I) appeared almost throughout the year, with the exception of January and the period of August and September. Thus, spawning appears to occur between February and July, and then again in October, the month in which the greatest proportion of spent individuals appeared.

[FIGURE 2 OMITTED]

The index of gonad maturity (IGM) increased from October to December, staying high for the rest of the year, but with smaller peaks in January, March, and August (Fig. 3). The gonad index (GI) showed March, June, and September as months with maximum gonad development (Fig. 4). Main reductions in the gonad index, suggesting spawning, were observed during October-November and March-April, and a smaller one in June (Fig. 4).

[FIGURES 3-4 OMITTED]

Recruitment

The recruitment pattern, estimated using the growth parameters L[infinity] = 163 mm and K = 0.2 [y.sup.-1] estimated by Hernandez (2001), shows that individuals entered the population throughout the year with a maximum in March (Fig. 5). This coincided with the period of maximum maturity, according to the proportion of individuals in Stage III and highest IGM (Figs. 2 and 3). Recruitment throughout the year coincides with the observation that mature individuals occur for the entire year, existing no defined seasonal pattern of reproduction. A small peak was observed in March.

[FIGURE 5 OMITTED]

Mean size at first maturity

According to Figure 6a, 50% of males reached sexual maturity at 52 mm shell height; such results fit the logistic model. It was thus estimated that all males were capable of reproduction at a size of 73 mm (Fig. 6a). Data for females also fitted the logistic model. Females became sexually mature shortly after the males at a shell height of 65 mm, with all females capable of reproduction at 77 mm (Fig. 6b). Spawned females 74-75 mm in height were identified in April.

[FIGURE 6 OMITTED]

Sex ratio

The sex ratio changed with age (Fig. 7). In the smallest size class (26.5 mm) females predominated, but only 8 individuals of that size class were examined. In the second analyzed size class (32.5 mm) males predominated and then tended to decline toward greater size classes (32.5-92.5 mm). In the greatest analyzed size class (98.5 mm) only females were found, but again, it was only a small sample (4 individuals) (Fig. 7).

[FIGURE 7 OMITTED]

Intersex individuals

Few individuals (13) could be classified as in the intersex phase, representing only 4% of the 325 individual examined. Individuals with both male and female characteristics had both a gonopore and an incipient penis similar to normal males, although microscopic examination confirmed they were females (masculinized females) with normal developed ovaries. These individuals measured 40-70 mm in shell height (Table I). Microscopic observations confirmed that some individuals with a gonopore were males (feminized males), and these were 55 to 73 mm in shell height presenting a normal developed testis. All these individuals were classified as in the intersex phase.

DISCUSSION

The coincidence between the major recruitment pulse and the maximum maturity, described by various methods (stages of maturity, IGM, GI) with also coincident results, suggest that the reproductive cycle described is reliable. While the peak of maturity occurs in the dry season, the presence of mature individuals throughout the year shows that variation between dry and rainy season seems to affect little, supporting a hypothesis of continuous reproduction and recruitment, a condition common in neotropical gastropods (Weber 1977). Rodriguez (1976) reported that in another area of the Caribbean, copulation by Melongena melongena occurred between December and July. These observations may have been biased by the methodology used by Rodriguez as they were based only on field observations where sampling days did not necessarily coincide with days when ovicapsules were present.

Continuous spawning, beginning at early age, represents a favorable condition for M. melongena, which is subjected to an artisanal fishery. According to growth data (Hernandez 2001), M. melongena becomes sexually mature in its third year (at sizes between 52 and 65 mm). Nevertheless, the fishery is capturing individuals as small as 12 mm shell height, the main captures occurring from >40 mm, with an average size of 57 mm (Hernandez 2001). Thus, most of the fished snails have not reproduced prior to capture. This is a situations which needs to be revised and regulated to prevent overfishing of this resource, affecting recruitment and thus population renewal. That is unless the fishery is inefficient, and a great proportion of individuals of all sizes escape capture.

As M. melongena appears to be gonochoric, the observation of intersex individuals generates doubts. The presence of such individuals confirms the observations of Rodriguez (1976), which, in agreement with the present study, reported masculinized females as occurring at sizes between 40 to 62 mm in shell height. For comparison, both feminized males and masculinized females were found in Cispata Bay, at a shell height range of between 40 and 73 mm.

The decrease in the number of males with increasing size suggests the occurrence of consecutive hermaphroditism. Dominance by females is however frequent in populations of gonochoric molluscs, and becomes accentuated with increasing age, eventually generated by differential mortality (Fretter & Graham 1962, Gibbs et al. 1987). It has been found in populations of Nucella lapillus, affected by imposex, that the males are more abundant than females, as the latter suffer heavy mortality (Gibbs & Bryan, 1986 and Gibbs et al. 1987). But the identification of intersex individuals also points to the occurrence of hermaphroditism. This may however, be due to other specific causes such as those cited by Reed (1993) who indicated that sexual alternation in Strombus gigas could be due to abnormalities in the sex chromosomes. Horiuguchi et al. (1994) established that in neogastropods the development of male sex organs in females (termed "imposex") was a broad-scale problem described for numerous species. Gibbs et al. (1987) established that the presence of masculinized females in Nassarius obsoletus and Nucella lapillus could be produced by exposure of these species to toxic compounds present in antifouling paints such as tributylene and TBT (tributyltin chloride). Exposure to these pollutants of females of these species destroys the oviduct, suppresses oogenesis, and results in the development of a testis. The females become unable to reproduce and the population declines as a result (Gibbs & Bryan 1986). According to Horiuguchi et al. (1994), the degree of imposex in Thais clavigera and T. bronni, as indicated by the length of the penis, provides a rough estimate of TBT levels in the environment.

Event though, the change in sexual proportion with size and the existence of few intersex individuals suggests the occurrence of protandric hermaphroditism in M. melongena, the evidence is weak. If the occurrence of protandric hermaphtoditism is true, however, the fishery for M. melongena may be interfering seriously with reproduction, as the individuals are not allowed to reach the larger sizes. This needs to be further studied, following over time the same individuals., to determine if changes in sexual proportion with size and intersex individuals are really evidence of protandric hermaphroditism, or result from other causes.

TABLE 1. Intersex individuals identified for each size
class interval of Melongena melongena.

 Size class      Feminized    Masculinized
interval (mm)      males        females

     40                            1
     43
     46                            1
     49                            2
     52
     55              2             3
     58              1
     61
     64
     67
     70              1             1
     73              1

    TOTAL            5             8

Figure 7. Sex ratio (%) of Melongena melongena in relation
to size in Cispata Bay. The x-axis shows the mean total
shell height of each size class (mm). Numbers above columns
represent the total number of individuals analyzed.

sexual proportion (%)

            Shell height (mm)

            males    females

 8    26,5
12    32,5
28    38,5
17    44,5
23    50,5
21    56,5
24    62,5
18    68,5
48    74,5
37    80,5
27    86,5
17    92,5
 4    98,5

Note: Table made from bar graph.

ACKNOWLEDGMENTS

We are grateful to Hanne Cogolio and Jesus Cantillo for help in field sampling, and to the snail collectors, especially "Tanque". We are also thankful for logistical support provided by Luz Marina Arias, Chief of the INPA office in Cordoba, and to Evila Jarava Ossa for her collaboration. Further thanks are extended to the personnel of the Amaya Research Station, to Marisol Romero of the U.Catolica del Norte, Coquimbo, for guidance in the histological interpretation of M. melongena gonads and to Piedad Victoria for review and edition of the Spanish text of this MS. We add final thanks to Adriana Zetina, Cristian Aycaguer and C. D'Asaro for sending reprints of their research which were important to our preparation of this MS. Two anonymous reviewers are acknowledged for their comments, which helped to improve the manuscript.

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SANDRA HERNANDEZ (1) AND WOLFGANG B STOTZ (2)*

* Corresponding author: [e-mail]

(1) Fundacion Humedales, Calle 97 No 21-42 Bogota, Colombia (2) Universidad Catolica del Notre, Casilla 117 Coquimbo, Chile, [e-mail]