Breeding cycle and early development of the keyhole limpet Fissurella nigra Lesson, 1831.

ABSTRACT In Chile, various species of keyhole limpet of the Fissurella genus constitute a multispecific fisheries activity. Nevertheless, management of these species is limited because of insufficient understanding of their reproductive biology. Fissurella nigra Lesson, 1831, is one of the species currently extracted by local fishermen in southern Chile.

The breeding cycle of F. nigra was studied through changes in the stages of gonad maturity using histological techniques, as well as monitoring the temporal variations in the gonadosomatic index (G.S.I.). Minimum maturity size, spawning induction, artificial fertilization and early larval development were also studied to obtain data on the biological background of this species that will facilitate management and enhance culture feasibility. Fissurella nigra is characterized by a unimodal reproductive cycle with one breeding season between October and December, coinciding with the period when gametes are spawned. Fertilization is external and mainly nocturnal spontaneous spawning occurred between August and November (most abundant in October and November) in adult organisms kept under laboratory conditions. Spawning was induced in October by combining thermal shock with desiccation. This method proved successful, provoking massive spawning. The gonad became visible in 50% of organisms within a size range of 26.0-35.9 mm. Early development occurs within the egg membrane and the veliger larval stage is reached approximately three days after fertilization.

KEY WORDS: breeding cycle, keyhole limpet, larval development, Fissurella

INTRODUCTION

Species of the Fissurella Bruguiere, 1789 genus, commonly known as "lapas" or "chapes," are among those gastropods frequently consumed by the coastal inhabitants of Chile (Huaquin et al. 1998) constituting a multispecific fisheries activity of growing commercial importance. Annual extraction of keyhole limpets over the last five years reached a maximum of 12,800 metric tons in 2004 and a minimum of 3,400 metric tons in 2000 (Sernapesca 2004).

Fissurella nigra Lesson, 1831, is one of the principle species of keyhole limpet caught in southern Chile (Bretos et al. 1988a, Bretos et al. 1988b). Of the 13 species of this genus found along the Chilean coastline, F. nigra and F. maxima are the largest (70-110 mm) and are classified among the large keyhole limpets (McLean 1984). This species is distributed along the Chilean coastline from Punta El Tabo (33[degrees]30'S) (McLean, 1984) to Navarino Island (55[degrees] 12'S) (Dell 1971). However, this species is most abundant between Concepcion and the island of Chiloe. They inhabit the mid to lower-intertidal zone, fixed to the underside of rocks (McLean 1984).

Fissurella species are gonochoristic (sexes are separate). There are no external sexually dimorphic features. The testis of the male is beige-colored and the ovary of females is bright green (McLean 1984). Knowledge about the breeding cycle of Chilean species of keyhole limpets is scarce. An approximation of some breeding parameters has been made indirectly based on calculations of the Gonadosomatic Index (GSI) in Fissurella maxima (Bretos et al. 1983), and F. nigra (Bretos et al. 1988b). Breeding cycle and gamete morphology have been studied in F. crassa (Huaquin et al. 1988). Early development was studied in F. picta after artificial fertilization (Gonzalez et al. 1999). The purpose of this research work is to study the reproductive biology of Fissurella nigra to implement appropriate management strategies and evaluate culture feasibility.

MATERIALS AND METHODS

Environmental Conditions

To determine the relationship between environmental conditions and breeding cycle, average monthly surface temperature of seawater in Metri bay (41[degrees]36' S; 72[degrees]43'W) was recorded over the last decade (1991-2000).

Gonadosomatic Index

During the period April 1994 to March 1995, between 15 and 20 adult specimens over 55 mm were collected manually from the intertidal area of Metri Bay at monthly intervals (Fig. 1). Maximum length, wet weight (excluding shell) and gonad weight were measured in each specimen, using a balance 0.01 g accuracy. The gonadosomatic index (GSI) was determined based on the following equation:

GSI = GW/SPW x 100

GSI, gonadosomatic index; GW, gonad weight; SPW soft parts with gonad weight.

[FIGURE 1 OMITTED]

Minimum Maturity Size and Sex Proportion

Fifty-six specimens of F. nigra ranging between 4.34 and 81.76 mm length, were collected in July 1996. These specimens were dissected to determine the stage of gonad development macroscopically, based on color and structure, according to the methodology described by Pena (1987). Sex proportion was determined monthly.

Breeding Cycle

Parallel to GSI sampling, histological analysis of the central portion of the female gonad was undertaken monthly in 15-20 adult specimens, between April 1994 and March 1995. This was achieved by microscopic analysis of the central portion of the female gonad, after performing routine histological procedures. The maturity scale used was proposed by Ward (1966) for F. barbadensis and considers four stages: neutral (N). developing (D), fully developed (FD) and spawning (S). Frequency of these sexual maturity stages was determined monthly. Sex proportion was calculated by considering the total number of specimens collected for GSI calculations.

Spawning Induction and Artificial Fertilization

Female and male specimens of Fissurella nigra with a shell length of over 50 mm were transferred to the laboratory and conditioned during the months of July and August (winter) 1994, in preparation for spontaneous spawning: conditioning was achieved by maintaining specimens in darkness at 12[degrees]C, and feeding ad libitum with the red alga Gracilaria chilensis. A combined method of thermal shock and desiccation was used to induce spawning every 15 days in September and October (spring), as follows: specimens were maintained in emersion for one hour at room temperature (16 [+ or -] 2[degrees]C) and subsequently at 30[degrees]C for 30 min; they were then transferred to aquaria and kept in permanent darkness with ad libitum feeding at a temperature of 12[degrees]C.

Gametes were also obtained by dissecting the gonads during periods of maximum maturity. The oocytes obtained by this method were rinsed for 10 min in filtered seawater (1 [micro]m). at 10[degrees]C. Once swelling of the gelatinous coat occurs, fertilization is carried out by adding a sperm suspension in a proportion of 15-17 per ovule (Gonzalez et al. 1999).

Embryonic and Larval Development

The fertilized eggs obtained as a result of spontaneous spawning, or through dissection of the gonad, were placed in filtered seawater in aquaria with a 6 L capacity, (1 [micro]m), at a temperature of 10[degrees]C, 32[per thousand] salinity, and kept in darkness with a permanent air supply. Once hatched, larvae were maintained in the same aquaria and conditions at a density of 1 larva/mL.

Statistical Analysis

To evaluate differences between monthly values of the Gonadosomatic Index, a one-way variance analysis (ANOVA) was used with an a posteriori Tukey test. Differences in sex proportion were established using the Student t test applied to the average value obtained during the study period. The statistical analysis system (SAS) was used.

RESULTS

Environmental Conditions and Gonadosomatic Index

The annual GSI curve is unimodal, and is inversely related to water temperature (Fig. 1). The GSI values were higher between May and August. During September average GSI decreased, remaining low until February. The neutral stage was not detected in any of the specimens analyzed. There were no significant statistical differences in the GSI values of June, July, and August, but GSI values did vary during the remainder of the period (Fig. 1, Table 1).

Breeding Cycle

Three of the four development stages described by Ward (1966) were identified in the female gonad of Fissurella nigra. The developing stage (D), when the gonad is characterized by thick follicular trabeculae and abundant, pear shaped primary oocytes, joined by a peduncle to the follicular wall, occurred mainly during April, May, June, September, and October with over 50% frequency. On the other hand, the fully developed stage (FD) when ovary follicles are filled with irregularly shaped oocytes and follicular walls are thin with sparse oogonia, and the spawning stage (S) when a considerable number of ovary follicles are empty or with only a few remaining oocytes, occurred frequently during the months of July, August and November 1994, and March 1995 (Fig. 2).

[FIGURE 2 OMITTED]

Minimum Maturity Size and Sex Proportion

Both sexes present visible gonad in 50% of organisms in the size range 26.0-35.9 mm length, (75% females and 25% males). Length of the smallest female was 10.87 mm whereas length of the smallest male was 34.75 mm. Average sex proportion for the entire sampling period was 1:1, with occasional monthly variations but no significant statistical differences (t = 3.24; df = 22; P = 0.005).

Spawning Induction, Artificial Fertilization, and Early Development

The combined method of temperature change and desiccation used during October gave positive results. Nevertheless, mainly nocturnal spontaneous spawnings were obtained between August and November in animals conditioned in the laboratory, being most abundant in October and November. The gametes were released into the environment through the apical aperture, indicating external fertilization. In the laboratory it was observed that some of the females spawned out of the water, at the edge of the tank; this behavior could indicate that spawning occurs during low tide.

The F. nigra oocyte (1593.4 [+ or -] 142.7 [micro]m diameter) is protected by a rigid membrane or corion (2248.2 [+ or -] 107.9 [micro]m diameter) that has a fairly conspicuous opening or micropyle to enable sperm penetration. It possesses an external gelatinous coat (3683.2 [+ or -] 172.7 [micro]m diameter). The sperm has a conical head 6.6 [+ or -] 0.07 [micro]m and a flagellum of 24.1 [+ or -] 1.2 [micro]m length.

Embryonic development up to the trocophore stage occurs within the egg membrane and corresponds to the typical development described for gastropod molluscs, with elimination of the two polar bodies after fertilization; segmentation is of the spiral type with the formation of micromeres and macromeres. The trocophore larva F. nigra has a maximum average length of 219.8 [+ or -] 17.5 [micro]m, and swims actively within the egg membrane. Average diameter of the membrane is 320.6 [+ or -] 8.6 [micro]m. The prototroch has abundant, elongated cilia and forms a crown towards the anterior end; granules of dark green vitellus can be observed at the posterior end of the larvae (Fig. 3a). The trocophore stage is reached around 48 h after fertilization and lasts approximately 24 h, during which period the larval shell is formed and the larvae break through the egg membrane, hatching in a veliger stage approximately 72 h after fertilization (Table 2). The gelatinous coat was not observed at this development stage. During the veliger stage, length is approximately 308.0 [+ or -] 10.3 [micro]m; they swim actively using a small velum with long cilium located at the anterior end. Traces of dark green vitellum were observed within the larval shell, at the posterior part (Fig. 3b). The opercule appears on day 2 after spawning. Larvae at the veliger stage remained alive for 72 h under the culture conditions previously described, without reaching metamorphosis.

[FIGURE 3 OMITTED]

DISCUSSION

The results obtained enable us to conclude that F. nigra has a unimodal breeding cycle with a maturation period in May, June, July, and August, during which no significant differences in the GSI values were detected. The breeding season spans the months of October, November, and December. The period of maximum maturity coincided with the lowest water temperatures, whereas the spawning season occurred when water temperature increased. Breeding behavior in relation to temperature is similar to that observed in F. maxima (Bretos et al. 1983) and F. crassa (Huaquin et al. 1998). As has been observed in F. nigra, gonad maturity is related to gradual changes in water temperature in accordance with Orton's law, which indicates that a gradual change in the water temperature induces gametogenesis, whereas a rapid change provokes spawning (Giese & Pearse 1974). The latter is confirmed by the positive results obtained in October from spawning inducement with changing temperatures, even if gametes were observed in the gonads throughout the study period. On the other hand, spawning would also appear to be related to the tidal cycle, given that some animals spawned at the edge of the aquaria in October and November, when spawning probably occurs in the natural environment (Figs. 1 and 2). The periods when mature animals were in the fully developed and spawning stages (50% to 80%) frequently coincided with the months when the highest GSI values were registered (Fig. 2).

The F. nigra oocyte is similar to that described for Tegula funebralis by Moran (1997); F. crassa by Huaquin et al. (1998), and Diadora aspera by Hadfield & Strathmann (1996). In the latter species, channels have been identified in the gelatinous cover to facilitate penetration of the sperm. This membrane swells during the breeding season of F. nigra, when spontaneous spawning occurs, becoming less dense and enabling penetration of the sperm. Given that fertilization is external, sperm correspond to the ecto-aquasperm type, as described by Jamieson (1987). Furthermore, the sperm morphology classifies it as a primitive species, according to Franzen (1956).

Minimum maturity size in F. nigra is calculated between approximately 25 and 30 mm length, less than that recorded by Bretos et al. (1988b) in specimens of the same species (4246 mm) from Queule (39[degrees]23'S; 73[degrees]13'W) 250 km north of Metri Bay. This value is in accordance with data obtained from studies of F. maxima by Bretos et al. (1983), who record the identification of ovaries and testicles in animals as small as 27.3 and 30.7 mm shell length, respectively.

So far, the most advanced stage described in F. picta is the free-swimming trocophore. This is reached 96 h after fertilization (Gonzalez et al. 1999). The time taken to reach each of the developmental stages does not differ greatly from that described for other species of gastropods. It has been established that in the northern hemisphere species, F. barbadensis, the planctonic larval period lasts three days (Lewis 1960) whereas in Diadora aspera hatching is produced between day 7 and day 8 after fertilization. The hatching process occurs in two stages, first the internal capsular membrane disappears, apparently caused by enzymatic action, and subsequently the larvae penetrate the gelatinous coat by mechanical action (Hadfield & Strathmann 1996). Metamorphosis was not reached in this study. However, it was observed that the veliger larvae still had drops of vitellum three days after hatching and remained on the bottom of the aquarium. Given this characteristic, it is probable that the initial larval stages of F. nigra are of the lecitotrophic type.

Based on the results obtained for Fissurella nigra, it can be concluded that its breeding characteristics are common to those of arqueogastropods and to other species of the Fissurella genus inhabiting the Chilean coast. Culture potential for F. nigra is good, given that it can be conditioned and maintained in the laboratory for long periods of time; it spawns spontaneously under artificial conditions and has a short larval development period. The larval stages do not require external feeding, which is an additional advantage. Nevertheless, the conditions necessary to ensure complete larval development, including metamorphosis, have still to be defined.

ACKNOWLEDGMENTS

The authors thank the Research Department of the Universidad de Los Lagos, DIULA No: 304.18 and Fondecyt, Project No: 040.93 for financing this study. The collaboration of Erick Baqueiro in the revision of the paper and Susan Angus in the translation of the manuscript is also gratefully acknowledged.

LITERATURE CITED

Bretos, M., L. Tesorieri & L. Alvarez. 1983. The biology of Fissurella maxima Sowerby (Mollusca: Archeogastropoda) in Northern Chile. 2. Notes on its reproduction. Biol. Bull. 165:559-568.

Bretos, M., J. Gutierrez & Z. Espinoza. 1988a. Estudios bioldgicos para el manejo de Fissurella picta. Medio Ambiente 9:28-34.

Bretos, M., V. Quintana & V. Ibarrola. 1988b. Bases biologicas para el manejo de Fissurella nigra. Medio Ambiente 9:55-62.

Dell, R. K. 1971. The marine mollusca of the Royal Society Expedition to Southern Chile, 1958-59. Records of the Dominion Museum (Wellington) 7:155-233.

Franzen, A. 1956. On spermiogenesis, morphology of spermatozoon, and biology of fertilization among invertebrates. Zool. Bidr. Uppsala 31:355-485.

Gonzalez, M. L., M. C. Perez, D. A. Lopez, J. M. Uribe & J. M. Castro. 1999. Early development in Fissurella picta (Gmelin). Veliger 42:275-277.

Giese, A. C. & J. S. Pearse. 1974. Introduction. In A. C. Giese and J. S. Pearse (eds.) Reproduction of Marine Invertebrates. New York: Academic Press. pp. 1-49.

Hadfield, M. G. & M. F. Strathmann. 1996. Variability, flexibility and plasticity in life histories of marine invertebrates. Oceanologica Acta 19:323-334.

Huaquin, L., R. Guerra & M. Bretos. 1998. Identificacion del sexo y morfologia de gametos de la lapa Fissurella crassa (Lamarck, 1822) (Mollusca: Archaeogastropoda). Rev. Biol. Mar. Oceanogr. 33:223239.

Jamieson, B. G. 1987. A biological classification of sperms types with special reference to annelids and molluscs, and an examples of spermiocladistics. In: H. Mohri, editor. Nez Horizons in sperm Cell Res. Tokyo: Japan Scientific Societies Press. pp. 311-332.

Lewis, J. B. 1960. The fauna of rocky shores of Barbados, West Indies. Can. J. Zool. 38:391-435.

McLean, J. H. 1984. Systematics of Fissurella in the Peruvian and Magellanic faunal provinces (Gastropoda: Prosobranchia). Natural History Museum, Los Angeles County, Contributions in Science 354:1-70.

Moran, A. L. 1997. Spawning and larval development of black turban snail Tegula funebralis (Prosobranchia: Trochidae). Mar. Biol. 128:107-114.

Pena, J. B. 1987. Reproduccion de Gasteropodos prosobranquios. En: Espinosa y Labarta, editors. Reproduccion en acuicultura. Plan de formacion de tecnicos superiores. Madrid Espana: Comision asesora de Investigacion Cientifica y Tecnologica.

SERNAPESCA. 2004. Anuario Estadistico de Pesca. pp. 194.

Ward, J. 1966. The breeding cycle of keyhole limpet Fissurella barbadensis Gmelin. Bull. Mar. Sci. 16:685-695.

MARGARITA C. PEREZ, * MARIA L. GONZALEZ AND DANIEL A. LOPEZ

Laboratorio de Cultivos Marinos. Departamento de Acuicultura y Recursos Acuaticos. Universidad de Los Lagos. Avda. Fuchslocher 1305. Osorno, Chile

* Corresponding author. E-mail: [e-mail]

TABLE 1.
ANOVA Table. Average monthly CSI in Fissurella nigra.

Source of         Mean of
Variation   df    Squares     F     Probability

Month       11     363.68   15.01     0.0001
Error       240     24.23

TABLE 2.
Embryonic and larval developmental stages of
Fissurrella nigra and time lapse after
fertilization.

                                 Time
                                 Lapse
                                After
                             Fertilization
Developmental Stage             (hours)

Gastrula                          24
Intra capsular "trocophore"       48
Free-swimming "veliger"           72