Bluefin tuna otolith ageing: Dr. Dave Secor’s Lab
March 12-14, 2012
Participants
Dr. Dave Secor
Ben Gahagan
Dheeraj Busawon
The purpose of this meeting was to:
-
Receive training on the operation of the micro-mill, in order to extract the core
region of the Bluefin tuna otolith for Chemical analyses (Annex 1: Micro-mill
protocol)
-
Discuss ageing issues (light type, placement of the 1st annulus, anchor point and
ageing section) identified in previous workshops.
1. Light type
In our previous workshops a topic of discussion was whether to use reflected or
transmitted light. In order to address this point, Ben Gahagan imaged a series of otoliths
(n = 31) using both lights. Dheeraj Busawon and Robert Allman aged the sections (see
BFT_Light_comparison_TvsR_DB & BFT_Light_comparison_TvsR_RA) and a paired
t-test was used to test for significant differences in ages between light types. In the case
of Dheeraj, the results showed that there was no significant difference in ages between
light types (t = 0.6427, p-value = 0.5253). However, a significant difference in ages
between light types was found for Robert (t = -3.1435, p-value = 0.003745) (Figure 1).
Further analyses showed that there are also significant differences in assigned
ages between readers using the same light type (Transmitted light Dheeraj vs. Robert: t =
-3.5998, p-value = 0.001132; Reflected light Dheeraj vs. Robert: t = -6.8449, p-value =
1.352e-07).
otoliths. Dheeraj Busawon (a) and Robert Allman (b).
N
N
N
C
C
_9
1
_8
2
_7
9
_6
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_1
10
_1
03
_1
00
C
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C
C
C
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C
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_3
0
C
C
C
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N
N
N
N
N
N
N
N
O
M
_7
_5
4
G
O
M
_7
_5
7
G
O
M
_7
_6
7
G
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M
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0
G
Image Quality
Transmitted - Reflected
N
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C
C
_9
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00
C
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C
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Image Quality
Transmitted - Reflected
a)
1.5
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0
-0.5
-1
-1.5
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-2.5
b)
3
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1
0
-1
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Figure 1. Age difference between reflected and transmitted light for 31 Bluefin tuna
2. 1st Annulus and Anchor point
The main point of discussion has been the placement of the 1st annulus based on
measurement and selecting an anchor point (see OtoInterpCriteriaReview.doc). During
this meeting we carried out measurements on yearlings and would like to propose the
following updates:
Anchor point: In our previous discussions, we had considered that the first
inflection point might be a landmark to identify the zone of growth through the first year
of life. However, through observation of images, it was noted by both Ben and I that the
first inflection of otolith sections is quite variable and might not be suitable as an anchor
point for accurate measurements of the 1st annulus. Therefore, we would like to propose a
new anchor point at the bottom centre of the bridge between the two arms (Figure 2).
Measurement for the 1st annulus based on the novel anchor point (Table 1): There
are two possible ways to measure the first annuli: Measure 1 is going up the inner ventral
arm and Measure 2 is going along outer ventral arm (Figure 2). On the basis of this novel
measurement methodology, we measured 8 yearling otoliths and propose a floor (1st
annulus can’t be any lower than this measurement) of 0.75 mm for Measure 1 and 1.14
mm for Measure 2. As the sample size is quite small it would be useful to obtain further
measurements from western Atlantic Bluefin tuna, in order to confirm this floor
measurement (Note: the measurement is very close to that proposed by Dr. RodriguezMarin for eastern Bluefin tuna (see OtoInterpCriteriaReview.doc).
a)
b)
Figure 2. Bluefin tuna otolith sections of known juveniles illustrating anchor point and
1st annulus measurement: Measure 1 (a) and Measure 2 (b).
Table 1. Measurement of 1st annulus based on novel anchor point.
ID
Measure 1 (mm)
Measure 2(mm)
2003_5
0.82
1.191
2003_15
0.936
1.396
2003_25
0.95
1.32
2004_1
0.755
1.184
2004_2
0.769
1.293
2004_4
0.903
1.53
2004_5
0.911
1.317
2004_7
0.758
1.147
Average
0.85025
1.29725
3. Ageing section
Furthermore, a point worth mentioning is the ageing section, as some labs use the V
shape sections and others the Y shape sections. Although, no significance difference in
counts of serial sections has been found within labs, Dr. Rodriguez–Marin noted that:
‘Comparing the otolith ventral arm length of the three sections by otolith, a decreasing trend was
found as the section is closer to the front of the otolith or rostrum. That means that ventral arm
are shorter in V shape sections in comparison with Y shape ones coming from the same sample’.
This would suggest that it may be important to standardize the ageing section. In previous
discussions, it was proposed that we could use the first otolith for micro-milling and the
second otolith for ageing. Due to the possibility of novel methodologies and repeatability
of analyses, it is important to preserve the second otolith. Therefore, the ageing and
micro-mill sections should be taken from the same otolith.
The micro-milling procedure developed by Dr. Secor is as follows (Figure 3):
1. Embed otolith sulcus side down.
2. Make first cut near the edge of the anti-rostrum (section should contain only a very
thin portion of the anti-rostrum).
3. Make a second cut 2 mm from the first cut towards the rostrum edge.
4. Mount the section rostrum side down.
5. Correct section will look like anti-rostrum is not connected to the rest of the otolith.
dge
al e
n
i
m
Termin
al edge
Sulcus Acusticus
ial do
Med
rsal r
idge
Ter
Sectioned area
Focus
Rostrum
st r
Po
ru
ost
dia
Me
l
al
ntr
ve
rid
ge
m
Figure 3. Bluefin otolith section for Micro-milling; black lines indicates where the
otolith is sectioned (section = 2 mm). Red line indicates the ageing section (V shaped)
used in Dr. Secor’s lab. Green line indicates alternative ageing section (Y shaped: used in
DFO and Spanish Labs).
Figure 3 illustrates the Micro-mill Bluefin otolith section: the section is 2 mm thick and is
micro milled (approximately 1.5 mm) from the post rostrum side, therefore leaving a 0.5
mm V section for ageing after polishing. On the basis that we found no significant
difference in age between adjacent serial sections and the assumption that both the V and
Y section are adjacent to the otolith core, the DFO lab proposes to adopt Dr. Secors
protocol (i.e use V section for ageing).
4. Next steps
The multiple section comparison (previous communications) and light type comparison
show inconsistencies of interpretation across the various labs. In order to address this
issue, the DFO lab proposes to circulate approx. 50 high quality (4 according to
readability scale) annotated images which would be further annotated by the various labs.
The aim of this exercise would to assess consistency of interpretations when using
identical criteria. We propose that the following protocol be used:
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Use the anchor point and floor measurement proposed above
-
Two annotated readings of each section per lab; annotation to include anchor
point and 1st annulus measurement (similar to Figure 2).
Note: These high quality images could be used for the reference set. Therefore, if labs
want to add to the distributed set, that would be a good starting point for our reference
collection.