Crabtree et al.: Reproduction of Albula vulpes 
457 
is also no published information on bonefish fe- 
cundity. In this article, we estimate the age and 
length at which sexual maturity is attained and 
describe the seasonal cycle of gonadal development 
in bonefish from waters off the Florida Keys. We 
also estimate the total fecundity of 33 bonefish 
collected from these waters. 
Methods 
Sampling 
We examined 528 bonefish collected from South 
Florida waters from February 1989 to April 1995. 
Most of these bonefish were caught with hook-and- 
line gear either by biologists or by a single profes- 
sional bonefish guide and his anglers from waters 
off the Florida Keys and in Florida and Biscayne 
Bays. Five bonefish caught with hook-and-line 
gear were obtained from taxidermists in Fort Lau- 
derdale and five others from tournaments in the 
Keys. Supplemental collections of small bonefish 
(<425 mm) were made with various-size seines and 
gill nets in waters off the Keys. Ages, based on vali- 
dated sectioned otoliths and growth rates of these 
bonefish, were described by Crabtree et al. (1996). 
Fork length (FL) was measured to the nearest 
millimeter (mm), and fish were weighed to the 
nearest gram. Sex, gonad condition, and gonad 
weight (g) were recorded. Gonad samples for his- 
tology and for estimation of fecundity were re- 
moved from the fish and preserved in 10% buff- 
ered formalin; they were later soaked in water for 
one hour and then stored in 70% ethanol. Histo- 
logical sections of gonads from 437 bonefish rang- 
ing from 228 to 702 mm were prepared and as- 
sessed for reproductive state. Gonad samples were 
processed histologically with a modification of the 
periodic acid Schiff’s (PAS) stain for glycol-meth- 
acrylate sections, with Weigert’s iron-hematoxy- 
lin as a nuclear stain and metanil yellow as a coun- 
terstain (Quintero-Hunter et al., 1991). 
Oocyte staging 
Oocytes were staged and counted from histological 
preparations at lOOx with a compound microscope 
attached to a digital image-processing system. Three 
oocyte stages were recognized in bonefish ovaries: 
primary growth, cortical alveolar, and vitellogenic 
(Wallace and Selman, 1981; Fig. 1A). In addition, we 
counted PAS-positive melanomacrophage centers 
(Ravaglia and Maggese, 1995), which were present 
in many ovaries (Fig. IB). When stained with the 
Figure 1 
(A) A histological section from an ovary of a 677-mm-FL bone- 
fish, Albula vulpes, showing oocyte stages. PG = primary 
growth oocytes, CA = cortical alveolar oocytes, and V = 
vitellogenic oocytes. Scale bar = 400 microns. (B) A histologi- 
cal section showing PAS-positive melanomacrophage centers 
(PAS), cortical alveolar oocytes, and primary growth stage 
oocytes in a regressed ovary from a 692-mm-FL bonefish. When 
stained with periodic acid Schiff’s stain, melanomacrophage 
centers are brilliant purple. Scale bar = 100 microns. (C) A 
histological section showing PAS-positive melanomacrophage 
centers in a regressed testis from a 586-mm-FL bonefish. Scale 
bar = 50 microns. 
PAS stain, these structures are brilliant purple. 
Melanomacrophage centers are thought to be active 
in degrading atretic oocytes, postovulatory follicles, 
and residual cells of the spermatogenic cycle (Chan 
et al., 1967; Ravaglia and Maggese, 1995). At least 
300 combined oocytes and melanomacrophage cen- 
ters per slide were staged and counted in arbitrarily 
chosen fields, and frequencies were expressed as a 
percentage of the total count. We counted all struc- 
tures that had at least 50% of their area visible in a 
field before moving to the next field. The presence of 
atretic hydrated oocytes was also noted. 
