Brule et a\ : Reproduction in Mycteroperca bonaa 



465 



from the small craft-fleet, whose crew captured them using 

 spear guns at depths ranging from 4 to 20 m, in areas close 

 to the port of Progreso, between November 1998 and May 

 1999. On Alacranes Reef, black groupers («=206) were cap- 

 tured with spear guns by small-craft fishermen at depths of 

 10-12 m between November 1999 and February 2000. 



Fork and standard lengths (FL, SL), whole and gutted 

 weights (WW, GW), and weight of gonads (gW), were re- 

 corded for all collected fish. All lengths reported in the pres- 

 ent study are fork length and all weights are gutted weight. 

 In discussion, total length data for Cuba (Garcia-Cagide 

 and Garcia, 1996) and Florida (Crabtree and Bullock, 

 1998) populations were converted to fork lengths by using 

 the fork-length to total-length relationship calculated by 

 Crabtree and Bullock ( 1998). 



Criteria presented by Sadovy and Shapiro (1987) were 

 used to diagnose sequential hermaphroditism in black grou- 

 per of Campeche Bank. Sex by size-frequency distributions 

 for black groupers were compared by using the Kolmogorov- 

 Smirnov nonparametric test, and differences between male 

 and female mean fork lengths were analyzed by using a 

 one-tailed 2-test (if n>30) or a one— tailed <-test (if n<30). The 

 male-to-female ratio (M:F), excluding transitional-stage 

 fish (referred to as "transitional fish" in this article), was 

 calculated and Pearson chi-square or Yates's corrected chi- 

 square goodness-of-fit tests were carried out to determine if 

 sex ratio differed significantly from unity (Scherrer, 1984). 

 Significance level, «. was 0.05 in all instances. 



Sex-dependent change in fin pigmentation, as described 

 by Crabtree and Bullock (1998), was examined in a sub- 

 set of fish (« = 104) caught from Alacranes Reef between 

 August and November 2000. The colors of the pectoral, 

 anal, dorsal, and caudal fins were recorded and histologi- 

 cal sections of the gonads were prepared and assessed for 

 sex identification. 



For all the fish sampled in all locations, sex and sexual 

 development were determined by examination of the micro- 

 scopic structure of the gonads. These were preserved in 

 Bouin's fluid, embedded in Paraplast and sectioned to 6 ftm 

 thickness. Ovaries and testes sections were stained in Gabe 

 and Martoja's triple stain for light microscopy (Gabe, 1968). 

 Fish were identified as female, male, or as transitional. 

 Based on red grouper microscopic features for oogenesis 

 (Brule et al., 1999) and for spermatogenesis (Moe, 1969), 

 six descriptive stages were recognized in black grouper 

 ovaries and five in the testes. Histological sections of the 

 ovaries were also scanned for the presence of postovulatory 

 follicles and atretic oocjrtes in alpha or beta stages (Lam- 

 bert, 1970). Using the criteria defined by Smith ( 1959) and 

 Sadovy and Shapiro ( 1987 1, we considered individuals with 

 gonads containing primarily ovarian tissue, degenerating 

 or not, with few clusters of spermatocytes, spermatids, or 

 spermatozoa to be undergoing sexual inversion. According 

 to the sexual classes defined by Brule et al. ( 1999) for red 

 grouper, female and male black grouper were classified as 

 resting, ripening, ripe-running, or spent, and fish in the 

 process of sexual inversion were classified as transitional. 

 Using the histological features considered by Shapiro et al. 

 (1993) as sign of prior spawning activity for the red hind 

 (Epinephelus guttatus) we were able to distinguish resting 



mature females from immature (virgin) females that had 

 never spawned. 



Reproduction periodicity was evaluated for both sexes 

 by examining seasonal variations in the gonadosomatic 

 index (GSI=100 xgW/GW) and in the relative proportion of 

 individuals in each sexual class. Specimens from offshore 

 waters taken during different years were pooled by month, 

 and mean GSI values and percent frequencies of sexual 

 classes were generated monthly for a single year Immature 

 individuals were discarded from this analysis. 



Size at which SO'X of females were sexually mature (L,;q) 

 was determined by using a binary logistic regression (SYS- 

 TAT statistical computer package for Windows, version 8.0, 

 SPSS Inc., Chicago, ID. For our analysis, resting mature, 

 ripening, ripe-running, and spent females were considered 

 as sexually mature individuals. Moreover, the minimum 

 size at which females become sexually mature (L^,;„) was 

 recorded, and the percentage of females of maximum 

 length at first maturity, ^^iJ^^nax with L^^^ ~ maximum 

 length of females recorded in samples, was determined 

 (Grimes, 1987). Sexual transition was analyzed by using a 

 binary logistic regression to estimate the length at which 

 50% of the females transformed to males (P^q) according to 

 Crabtree and Bullock (1998). The size range and median 

 size at which sex inversion occurs were estimated by fol- 

 lowing the procedures of Shapiro ( 1984). Furthermore, the 

 variation in size at sex change was analyzed by using two 

 ratios defined by Shapiro (1987): ratio 1, size range of tran- 

 sitional fish divided by maximum size of fish in samples; 

 and ratio 2, range of overlap in size of males and females 

 divided by maximum size of fish in samples. 



Results 



Size-frequency distributions 



All individuals collected from inshore waters were females 

 ranging from 25.6 to 58.0 cm in length (Fig. 2). The off- 

 shore fish sample, which did not include the Alacranes Reef 

 sample, was composed of 75.1% females, 24.3% males, and 

 0.6% transitional fish. Females ranged in size from 57.0 to 

 123.5 cm, males from 86.0 to 132.0 cm, and transitional fish 

 from 99.0 to 121.5 cm. The Alacranes Reef sample was com- 

 posed of 95% females ranging in size from 46.0 to 100.0 cm 

 and 5% males from 97.0 to 135.0 cm. In the offshore sample, 

 the male size range differed significantly from that of the 

 females (Kolmogorov-Smirnov; 7!=875; P<0.05), and male 

 mean fork length (114.6 ±7.1 cm; mean ±SD) was greater 

 than female mean fork length (96.6 ±12.1 cm; one-tailed 

 2-test, n=875; P<0.05). Similar results for male and female 

 size ranges (Kolmogorov-Smirnov; n=206;P<0.05) and male 

 (115.7 ±12.9 cm) and female (67.6 ±11.2 cm) mean fork 

 lengths (one-tailed t-test, «=206; P<0.05) were observed for 

 black grouper from the Alacranes Reef sample. 



Sex ratio 



The male-to-female ratios were calculated for each 5-cm 

 size class from 25.1 to 135.0 cm length (Table 1). The 



