170 
Fishery Bulletin 119(2-3) 
Otolith preparation and age determination 
The otolith corresponding to the blind side of each Indian 
halibut (i.e., the side of the fish facing downward and 
therefore having no eyes) was selected for aging purposes 
because it has been proven that the blind-side otolith in 
some flatfishes provides a clearer interpretation of growth 
zones than the eye-side otolith (Lear and Pitt, 1975; 
Forsberg’; Armsworthy and Campana, 2010). The whole 
blind-side otoliths of 272 fish were each placed in a black 
dish with low viscosity immersion oil, with the ventral side 
facing upward, and photographed under reflected light by 
using a Leica DFC 425 camera (Leica Microsystems Inc.®, 
Buffalo Grove, IL) mounted on a Leica MZ7.5 dissecting 
microscope (Leica Microsystems Inc.). Captured images 
were viewed by using Leica Application Suite, vers. 4.3 
(Leica Microsystems Inc.), enabling opaque zones in each 
otolith to be marked and counted. The number of opaque 
zones in the whole blind-side otoliths were counted on a 
single occasion by the primary reader (senior author). 
Those same otoliths were cleaned, dried, and embed- 
ded in a clear epoxy resin, along with the blind-side oto- 
liths from another 265 individuals, and cut transversely 
through the primordium into sections of ~250-300 pm 
with a low-speed diamond saw (IsoMet Low Speed Preci- 
sion Cutter, Buehler Ltd., Lake Bluff, IL). Otolith sections 
° Mention of trade names or commercial companies is for identi- 
fication purposes only and does not imply endorsement by the 
National Marine Fisheries Service, NOAA. 
Figure 1 
Photographs of the (A and C) whole otoliths and (B and D) corresponding transverse sections of the blind 
side of otoliths of 2 Indian halibut (Psettodes erumei) caught in the Indian Ocean off the Pilbara coast 
in northwestern Australia during February and July 2014. Each photograph shows the dorsal edge (D), 
ventral edge (V), sulcus (S), and nucleus (N) of the otolith. The horizontal dashed line across each whole 
otolith indicates the position of the corresponding transverse section taken from each otolith. Panels C 
and D show 1 and 8 opaque zones (white circles), respectively. The location of the white circles reflects the 
axis along which opaque zones were counted. All scale bars are equal to 1 mm. 
were then mounted on microscope slides by using DePeX 
mounting adhesive (VWR International LLC, Radnor, PA) 
and a coverslip. Digital images of each sectioned otolith 
were taken, by using a 12-MP Olympus DP70 camera 
(Olympus Corp., Tokyo, Japan) mounted on an Olympus 
BX51 compound microscope (Olympus Corp.) and by 
employing transmitted light. 
Counting of opaque zones on the digital images of sec- 
tioned otoliths was facilitated by the use of Leica Appli- 
cation Suite, which enabled such zones to be marked 
and readily counted and the distances required for mar- 
ginal increment analysis to be measured with confidence 
(Coulson et al., 2021). Opaque zones on the edge of the 
dorsal side of the otolith section, closest to the sulcus, 
were counted (Fig. 1). Measurements for marginal incre- 
ment analysis were recorded to the nearest 0.01 mm and 
taken on an axis perpendicular to the opaque zones on 
the dorsal side. 
To validate that a single opaque zone is formed annu- 
ally in the otoliths of Indian halibut, we analyzed trends 
observed throughout the year by using the mean monthly 
marginal increments (MI) on sectioned otoliths (i.e., the 
distance between the outer edge of the single or outermost 
opaque zone and the otolith periphery). When one opaque 
zone is present, the MI was expressed as a proportion 
of the distance between the primordium and the outer 
edge of the single opaque zone. When 2 or more opaque 
zones were present, the MI was expressed as a proportion 
of the distance between the outer edges of the 2 outer- 
most opaque zones. An approach based on the methods 
