Colmenero et al.: Reproductive biology of Lophius budegassa in the northwestern Mediterranean Sea 
393 
Cross sections, each 3-4 pm thick, were made with a 
manual microtome Leica Reichert-Jung 2040 (Leica 
Microsystems, 2 Wetzlar, Germany), stained with Lee’s 
stain (methylene blue and basic fuchsin), and mounted 
in a synthetic resin of dibutyl phthalate xylene (DPX) 
on microscope slides. Gonads were classified according 
to the morphological features and the presence of spe- 
cific inclusions (oil droplets, yolk granules, yolk vesi- 
cles, or postovulatory follicles) (Wallace and Selman, 
1981). The ovarian and testicular phases were defined 
by the developmental stage of the most advanced cell 
within the gonad (Yoneda et al., 1998b). 
Spawning season and size at first maturity 
The spawning season was established from the analy- 
sis of the monthly variation of the maturity phases and 
the changes in gonadosomatic (GSI) and hepatosomat- 
ic (HSI) indices for each sex (Afonso-Dias and Hislop, 
1996). Because immature specimens were not consid- 
ered, 1437 males and 1167 females were used to deter- 
mine both indices, which were calculated according to 
Yoneda et al. (2001) with the following equations: 
GSI = (GNW / GW) x 100. ( 1) 
HSI = ( LW / GW) x 100. (2) 
Size at first maturity (L50) was determined through the 
examination of males and females in mature phases 
(phase III, phase IV, or phase V) and immature indi- 
viduals collected during the spawning period (Duarte 
et al., 2001). Total length of all individuals was used 
to estimate L50, defined as the size at which 50% of all 
fish sampled were at sexually mature phases. Maturity 
curves were determined with a logistic curve (Pope et 
al., 1975): 
P = 100 / (1 + exp [a + 6TL]), (3) 
where P = the percentage of mature individuals as a 
function of size class (TL); and 
a and b = specific parameters that can change during 
the life cycle. 
A logarithmic transformation was applied to this equa- 
tion to calculate the parameters 0 and b by means of 
linear regression. 
Reproductive strategy and fecundity 
Patterns of ovarian organization and fecundity were 
tested by oocyte size-frequency distributions (West, 
1990). For our analysis, 36 fish, with lengths between 
20.0 and 72.5 cm TL, were randomly selected from 
all maturity phases. From these fish, 4428 oocytes — 
with more than 300 oocytes from each maturity phase 
(1=961; 11=1106; 111=1046; IV=381; V=934) — were mea- 
2 Mention of trade names or commercial companies is for iden- 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA. 
sured for their diameter with an image analysis pro- 
gram (Image-Pro Plus, vers. 5.0, Media Cybernetics, 
Inc., Rockville, MD) in combination with an Axioskop 
2 Plus microscope (Carl Zeiss Microscopy, LLC, Thorn- 
wood, NY) and a ProgRes C14 digital microscope cam- 
era (Jenoptik AG, Jena, Germany). Diameters were 
measured to the nearest 0.01 pm. The mean oocyte 
diameter by developmental stage was determined by 
calculating the diameter of all oocytes encountered in 
each subsample. Measurements were taken only of oo- 
cytes that were sectioned through the nucleus (Afonso- 
Dias and Hislop, 1996). 
Before fecundity was estimated, the gonads of 7 in- 
dividuals were divided into 3 sections (anterior, middle, 
and posterior) to test differences in mean oocyte den- 
sity within the ovary by using a one-way analysis of 
variance (ANOVA). This use of 3 sections ensured that 
the analyzed subsample represented the entire ovary 
(Murua et al., 2003). Batch fecundity (BF), the total 
number of mature eggs produced in a single spawn- 
ing batch by an individual female, was estimated by 
using the gravimetric method on the basis of the rela- 
tion between ovary weight and the density of oocytes in 
the ovary (Hunter and Goldberg, 1980). Three ovarian 
tissue samples of known weight, representing 10% of 
the total ovarian weight, were extracted from different 
areas of the same ovary (anterior, middle, and posterior 
ovarian lobe). These subsamples were collected from 15 
specimens with ovaries in phases III and IV with nei- 
ther postovulatory follicles nor atretic oocytes present. 
Because the oocytes could not be extracted from their 
mucogelatinous matrix without destroying them, whole 
tissue subsamples were mounted on several slides for 
analysis and covered with a cover slip. 
Images of each ovarian tissue sample were taken 
with a Canon Powershot SD870 IS digital camera 
(Canon USA, Melville, NY), and oocytes were counted 
manually with Image-Pro Plus software. Fecundity val- 
ues were obtained by examining Black Anglerfish with 
total lengths of 46-65 cm, TW of 1096-5592 g, GW of 
986-3600 g, and GNW of 88.70-2300 g. Batch fecun- 
dity for each female was calculated as a product of the 
number of secondary vitellogenic oocytes per unit of 
weight multiplied by the total ovarian weight (Yoneda 
et al., 2001). Relative batch fecundity (RBF), the total 
number of mature eggs released by a female during 
the spawning batch per gram of body weight of gutted 
fish, was calculated as BF divided by GW (Pavlov et 
al., 2009): 
BF = ( oocyte number / sampled GNW) 
x total GNW. (4) 
RBF = BF / GW. (5) 
Linear regression analysis was used to examine 
the relationships between BF and fish TL, TW T , and 
GW (Armstrong et al., 1992). Linear regression analy- 
sis also was applied to analyze the relationship be- 
tween RBF and TL. Mean potential fecundity was 
