Egg and early larval development of laboratory reared dusky grouper , Epinephelus marginatus ( Lowe , 1834 ) ( Picies , Serranidae ) *

The dusky grouper, Epinephelus marginatus (Lowe, 1834), is distributed throughout the Mediterranean Sea, and in the Atlantic, from the coast of the British Isles to Southern Africa, and along the coast of Southern Brazil. It inhabits rocky bottoms, from shallow waters to depths of 50 meters and lives in caves of stone blocks, where it spends most of its time (Jardas, 1996). The early life history is unknown, especially that describing egg and larval ecology, and it is difficult to determine these stages in ecological investigations. In Southeast Adriatic waters the dusky grouper spawns during late August and early September. Eggs were collected at the beginning of September, excluding larval stages (Skaramuca et al., 1989). Discoveries recording ripe eggs sites are rare, based on ichthyoplanktonic research (Sparta, 1935). In recent times, spawned eggs have been described (Barnabe, 1974 : Zabala et al., 1997). Little data whatsoever are available regarding larval stages, except for the description by Barnabe (1974), and one picture of larva published later (Zabala et al., 1997). For the systematic study of larval abundance in population estimates, the identification of early stages is critical. This paper presents results of egg and embryonic development, including description of early larval stages, for laboratory spawned and reared dusky grouper. SCI. MAR., 62 (4): 373-378 SCIENTIA MARINA 1998


INTRODUCTION
The dusky grouper, Epinephelus marginatus (Lowe, 1834), is distributed throughout the Mediterranean Sea, and in the Atlantic, from the coast of the British Isles to Southern Africa, and along the coast of Southern Brazil.It inhabits rocky bottoms, from shallow waters to depths of 50 meters and lives in caves of stone blocks, where it spends most of its time (Jardas, 1996).The early life history is unknown, especially that describing egg and larval ecology, and it is difficult to determine these stages in ecological investigations.
In Southeast Adriatic waters the dusky grouper spawns during late August and early September.Eggs were collected at the beginning of September, excluding larval stages (Skaramuca et al., 1989).Discoveries recording ripe eggs sites are rare, based on ichthyoplanktonic research (Sparta, 1935).In recent times, spawned eggs have been described (Barnabe, 1974: Zabala et al., 1997).Little data whatsoever are available regarding larval stages, except for the description by Barnabe (1974), and one picture of larva published later (Zabala et al., 1997).
For the systematic study of larval abundance in population estimates, the identification of early stages is critical.This paper presents results of egg and embryonic development, including description of early larval stages, for laboratory spawned and reared dusky grouper.

MATERIALS AND METHODS
Broodstock were collected from southeastern Adriatic waters and were held from one to ten years in aquarium conditions, at ambient seawater temperatures (12-24°C) and salinities (36-38 ppt), pumped at eight meter depths and 30 meters distance from the shore.The fish were spawned using hormonal treatment (Glamuzina et al., 1998a) and eggs were fertilised with sperm from sex-reversed males (Glamuzina et al., 1998b).Dry fertilisation lasted for 15 minutes and the remaining spermatozoa were rinsed through a 350 µm sieve with a light spout of fresh seawater.The rinsed eggs were transferred to a glass jar and floating eggs were collected.Eggs and larvae were incubated at 23°C , flow-through sea water, with aeration from the bottom.Samples of 30 eggs were taken every hour, and samples of 30 larvae every six hours, for description and measurement, using an ocular microscope.Careful examinations were carried out, supported by photography and drawings.Later on, the samples were fixed in 8% buffered formalin for more detailed morphological studies.
The characteristics of newly-spawned ripe and fertilised eggs were noted, together with the duration of each embryonic stage.Embryogenesis characteristics were monitored.
Larval development was described using measurements of total length: the distance along the midline of the body from the tip of the snout to the end of caudal fin rays; standard length: the distance along the midline of the body from the tip of the snout to the end of the urostyle; preanal distance: the distance along the midline of the body from the tip of the snout to the anus; head length : the distance between the tip of the upper jaw and the cleithrum; body depth: the perpendicular depth of the trunk at the anus ; greatest body depth: body depth as its widest point; eye diameter, longer diameter of the yolk sac and diameter of oil globule.

Egg characteristics and embryonic development
The average egg diameter of the dusky grouper was 846.68 ± 41 µm, with sizes varying from 738-940 µm.However, within one hour following fertilisation, the egg diameter became more uniform.Samples of surviving eggs showed that there were no eggs diameters less than 840 µm and egg size increased to an average size of 869.6 ± 35.1 µm and remained so up to the hatching of larvae.
The eggs were transparent and spherical.Developing eggs had only one oil globule.Those of poorer quality were slightly opaque and had two or more oil globules.The eggs were buoyant, with oil globules having an average diameter of 160 ± 21 µm (range 115-220 µm).Shortly after stopping aeration in the tank, all eggs grouped at the water's surface.
One hour and five minutes after dry fertilisation, the blastodisk divided into two cells for the first time.Eggs developed in a manner typical for teleosteans (Ahlstrom and Ball, 1954).Table 1 details the next key phases of embryonic development.Figure 1 presents photographs of eggs.

Larval development
The average net total length of newly-hatched, dusky grouper larvae was 1.52 ± 0.07 mm.Larvae varied from 1.40-1.67mm in total length, and were characterized by huge yolk sacs, along almost the entire body, except for the small tail part (Figs.2a and 3 a).The body was somewhat curved around the yolk sac.After hatching, larvae floated in the water column, without significant movement, except for sporadic tail thrusts.If aeration of the tank was stopped, all larvae rose to the surface and collected in large formations.
A few hours after hatching, the larvae showed significant growth.The growth rate was highest during the first 24 hour, afterwhich it decreased significantly.Table 2 shows changes in all measured characteristics of the larvae during the first five days.Drawings and photographs of the larvae during this period are presented in Figures 2 and 3.
The appearance of two areas of pigmentation represents the basic morphological characteristic of the dusky grouper in its early larval stage.The first area, located in the middle between the anus and above and bellow the posterior edge of the notochord appeared on the second day following hatching (Fig. 2d).During the next few days, these two areas enlarged and finaly joined one another.A second area of pigmentation was above the front intestine and stomach.Pigmentation occurred here a day later and in a few days EGG AND EARLY LARVAE OF DUSKY GROUPER 375  became so intense that it covered the entire top area of the front intestine and stomach, totally hiding the swimbladder which started to develop (Fig. 2e).During the first five days of development, there was no visible pigmentation to be seen on any other area of the body.All larvae examined during the first five days of development were characterized by these two fields of pigmentation.Figures 2 and 3 show the morphological development of larvae in detail.
The mouth opened after 72 hours, becoming fully functional after 96 hours when larvae started to feed.The mouth opening was between 250-300 µm.

DISCUSSION
The egg size of dusky grouper described by other authors is significantly smaller, ranging from 0.67 mm for unfertilized and unhydrated eggs in Spanish waters (Zabala et al., 1997), to 0.75 mm for fertilised eggs in Andalusian waters (Barnabe, 1974).The eggs from southeastern Adriatic waters, as described by Skaramuca et al.(1989) with a diameter of 836 µm, were of a comparable size to those obtained in our experiments.But, the eggs obtained in captivity in Southern Italy were bigger with a diameter of 888 µm (Spedicato et al., 1995) The eggs of the dusky grouper, Epinephelus marginatus are among the smallest eggs described up to now for the genus.Such a small egg was reported for the camouflage grouper Epinephelus polyphekadion from the Red Sea waters with a average diam-eter of 757.3 µm (James et al., 1997) and for the marbled grouper Epinephelus microdon from Micronesian waters with a range from 769-832 µm (Tamaru et al., 1996).The egg size of other groupers is bigger (Tucker, 1991;Watanabe et al., 1995;Chen, 1990;etc.).There is no other data on egg size for other species of the genus Epinephelus in Mediterranean waters, especially for the Adriatic.
The same situation exists with the net size of newly-hatched larvae, as dusky grouper larvae are the smallest of those described in this genus.For Mediterranean species, there is no recorded data available on sizes of larval stages.
However, most other characteristics, including large yolk sac, head and body shapes, location of oil globules, short intestine tracts and especially, characteristic pigmentation, are almost identical for most of the species described in this genus.The areas showing pigmentation in the early developmental stages, above the digestive system and between the anus and the end of the notochord, are also noted in the species E. tauvina (Hussain and Higguchi, 1980), E. striatus (Powell and Tucker, 1992), as well as in E. fuscoguttatus (Kohno et al., 1993).Alongside the general similarities of the larvae, this offers the best morphological characteristic used in separating early grouper larvae from other fish.Problems can arise if the spawning season of more than one species of the genus Epinephelus overlaps, for example, the species Epinephelus costae spawning season in Adriatic coincides with the spawning of E. marginatus (Jardas, 1996).Additional research on this species, now in progress, should offer a solution to this situation.

FIG. 1
FIG. 1. -Pictures of dusky grouper eggs at different stages of embryogenesis: a) two-cell stage, b) gastrula, and c) embryo.

TABLE 2 .
-Changes in lengths and shape of the dusky grouper, Epinephelus marginatus yolk sac larvae during the first five days from hatching at constant temperature of 23°C.