Parkinson Booth and Lee 2012-

Journal of Fish Biology (2012) 80, 698–704

doi:10.1111/j.1095-8649.2011.03194.x, available online at wileyonlinelibrary.com

Validation of otolith daily increment formation

for two temperate syngnathid fishes: the pipefishes

Stigmatopora argus and Stigmatopora nigra

K. L. Parkinson*, D. J. Booth and J. E. Lee

University of Technology Sydney, School of the Environment, P. O. BOX 123, Broadway,NSW 2007, Australia

(Received 21 December 2010, Accepted 23 November 2011)

Otoliths were used for the first time to successfully validate the age of members of the family

Syngnathidae: the spotted pipefish Stigmatopora argus and the wide-bodied pipefish Stigmatopora

nigra. Otolith increments were deposited daily in (1) known-age juveniles ranging in age from 0 to

31 days and (2) adults that had been stained with alizarin complexone, and a hatch mark was found

on all otoliths which represented day 0. Otolith increment validation will allow development of

growth models for S. argus and S. nigra, essential to understanding and managing these exclusive

seagrass species. © 2012 The Authors

Journal of Fish Biology © 2012 The Fisheries Society of the British Isles

Key words: daily growth increments; otolith age validation; Syngnathidae.

Members of the family Syngnathidae (including seahorses, weedy seadragons and

pipefishes) are considered to be important flagship species of seagrass ecosystems

and are expected to be sensitive to changes in seagrass cover and composition (Fos-

ter & Vincent, 2004; Shokri et al., 2009). Hundreds of fish species use seagrass

meadows in eastern Australia as a juvenile nursery ground (Hoese, 1978) and while

some are commercially important (McNeill et al., 1992), others are numerically dom-

inant, such as the spotted pipefish Stigmatopora argus (Richardson 1840) and the

wide-bodied pipefish Stigmatopora nigra Kaup 1856. The effects of direct and indi-

rect disturbances to seagrass meadows, due to shoreline development and associated

changes in sediment load (Duarte, 2002), are the biggest threat to this genus of Syn-

gnathidae, as they are found almost exclusively within seagrass habitat in Australia

(Hoese, 1978; Connolly, 1994; Vincent, 1996).

Effective conservation and management of any fish species requires knowledge

of certain parameters of their life history and population demography (Beamish

& McFarlane, 1983). References to age and longevity are sparse within the syng-

nathid family and limited for S. argus and S. nigra (Duque-Portugal, 1989; Kendrick,

*Author to whom correspondence should be addressed. Tel.: +61 2 9514 8346; email: Kerryn.

[email protected]

698© 2012 The Authors

Journal of Fish Biology © 2012 The Fisheries Society of the British Isles

AG E VA L I DAT I O N I N P I P E F I S H E S 699

2002). Several studies have met with little success in attempting to use otolith incre-

ment width to age members of the Syngnathidae. For example, the otoliths of the

double-ended pipehorse Syngnathoides biaculeatus (Bloch 1785) were examined by

Takahashi (2000), and the otoliths of the hedgehog seahorse Hippocampus spinosis-

simus Weber 1913 by Do et al. (2006). These research programmes described the

otolith morphology within both syngnathid species, but were unable to discern any

otolith increments and therefore were unable to use otoliths as a tool to examine

growth and age of these species.

This study aimed to determine whether, in S. argus and S. nigra, otolith growth

increments are deposited daily and therefore can be used as accurate chronometers

in population demographic studies. To facilitate this, the otolith microstructure was

analysed, described and interpreted for S. argus and S. nigra in order to validate the

initial occurrence and periodicity of increment deposition. Otoliths from both species

were examined in newborn individuals, in known-age juveniles and in adults.

Stigmatopora argus and S. nigra were collected monthly from seagrass mead-

ows between January 2001 and December 2002 from Botany Bay (33 o57·39′ S;

151 o11·81′ E) and May and September 2008 from Kurnell, NSW, Australia

(33 o57·39′ S; 151 o11·81′ E). Newborn S. nigra were collected in situ after a male

gave birth in holding tubs in the field, while S. argus newborns were collected directly

from the seine and identified in situ in the field according to the taxonomic key in

Dawson (1980). Both species were anaesthetized in an ice slurry and preserved in

90% alcohol. Adult males of both S. argus and S. nigra species gave birth to young

in aquaria between May and September 2008. These young were kept in aquaria for

a period between 3 and 31 days after which they were anaesthetized and frozen for

further examination. Adult S. argus and S. nigra were collected in situ for captive

investigation of daily increment formation. Specimens were identified to species and

total length (LT) measured. In addition, to establish a measure of the minimum total

length (LTmin) for mature males, the brooding status of males was noted (i.e. the

presence or absence of an occupied brood pouch). This measure allows the repro-

ductive status of the population to be assessed without the need for dissection of

reproductive structures (Steffe et al., 1989). All specimens were transported alive in

aerated containers to aquaria at the University of Technology, Sydney.

Aquaria were assembled to mimic syngnathid habitat using artificial seagrass

mats, with temperature, pH and dissolved oxygen monitored every second day for

the duration of the experiment. Tanks were subjected to a natural photoperiod

(33◦

57′ S) and syngnathids were fed daily on Artemia sp. enriched with Super

SELCO (www.inve.com). A total of 21 syngnathids (seven in each tank), a combi-

nation of S. argus and S. nigra, were allowed to acclimate for 7 days before being

immersed in a solution of alizarin complex one [(AC), (alizarin-3-methylamine-N,N-

diacetic acid, C18H15NO8)]. They were immersed into solutions of 0 (control), 60 or

80 mg l−1 AC for between 4 and 6 h. Fish were held for 7 days post-staining and then

re-stained (as above) and kept alive for a further 8 days. All individuals were then

sacrificed and otoliths extracted, rinsed with water to remove any remaining tissue

and secured onto microscope slides using Crystalbond (www.crystalbond.com). All

dissected otoliths were kept in lightproof slide boxes to reduce any photodeterioration

until analysis by UV light microscopy (using appropriate filters to provide excitation

wavelength 490 µm) and environmental scanning electron microscope (ESEM).

© 2012 The Authors

Journal of Fish Biology © 2012 The Fisheries Society of the British Isles, Journal of Fish Biology 2012, 80, 698–704

700 K . L . PA R K I N S O N E T A L .

Initial examination of unpolished whole sagittal otoliths with a compound light

microscope revealed concentric growth increments, but without sufficient detail for

accurate counting. Due to the small size of the otoliths (mean diameter 100 µm), all

further examination was made using an ESEM.

Otoliths were prepared for ESEM imaging by grinding to the primordium using

3 µm lapping film; wet polishing using a PMT 08A-10 multiflex 20·32 cm (8′′) diam-

eter polishing cloth (www.flatlap.co.uk) and 0·02–0·06 µm colloidal silica (www.stru

ers.com) on a Kent 3-automatic polishing unit (www.kemet.com.au), then etched with

5% solution of ethylene diamine tetra-acetic acid (EDTA). Each otolith was exam-

ined using a Philips XL 30 ESEM (www.fei.com). The otoliths were viewed in WET

mode, using backscatter electrons (BSE) at 15 kV. Spot size varied between 4·5 and

5·5 and pressure between 0·4 and 0·8 torr. Otoliths were examined for evidence

of daily growth and for any obvious stress marks that may have deposited due to

handling the fishes.

When first examined after birth, sagittal otoliths of S. argus and S. nigra were c.

100 (n = 6; mean = 98·87 µm) and 50 µm (n = 3; mean = 45·04 µm) in diameter,

respectively. As they grew, a distinct darker increment or check mark appeared on

the otolith at this distance for both S. argus and S. nigra (Fig. 1), which was prob-

ably a hatch mark. Examination of these newborn juvenile specimens indicated that

increments were visible inside this hatch mark, but were subsequently considered to

be formed prior to birth, when developing in the pouch of the male adult. All spec-

imens from newborn juvenile to adult of both species exhibited this hatch mark on

Acc.V Spot Magn15·0 kV 4·5 3100x

Det WDBSE 9·8 0·9 Torr

20 µm

Fig. 1. Polished transverse section (ESEM micrograph) from the sagittal otolith of a Stigmatopora nigra

male (69 mm total length, LT). Note indicates hatch mark, while indicate interpretable daily

increments.

© 2012 The Authors



the otolith at a consistently different diameter for each species (S. argus c. 100 µm;

S. nigra c. 50 µm).

Assuming this hatch mark represents day 0, known-age juveniles of S. argus

showed increment numbers equal to number of days alive; y = 0·99x− 0·13; r2 =

0·99; n = 24, where y = expected age and x = increment count. The slope of the

overall regression (0·99 increments day−1) was not significantly different from 1

(t = 0·742; P< 0·001), thus it was concluded that increments were deposited daily

(Fig. 2). For S. nigra, the expected age of the juveniles was best described by the

equation y = 0·85x+ 3·3; r2 = 0·98; n = 6, while the slope of the overall regression

was not significantly different from 1 (S. nigra, t = 0·007, P < 0·001; Fig. 2), thus it

was concluded that increments were deposited daily in S. nigra juvenile specimens.

Low sample size for S. nigra means results for this species must be accepted with

caution.

For adults, 13 specimens were examined. Five immersed in 80 mg l−1 AC pro-

duced interpretable fluorescent daily increments (Table I; note that otoliths from fish

stained in AC concentration of 60 mg l−1 did not show evidence of stain), while

eight stress specimens produced notable daily increments that clearly corresponded

to the number of increments expected between stress events during the experimen-

tation period. Events were (1) collection from seagrass meadows and transportation

to laboratory, (2) first treatment date and (3) second treatment date. Each of these

events was separated by 7 days. For either species, the difference between actual

and expected increments clearly indicated daily growth (Table I).

Previous studies attempting to analyse the microstructure of syngnathid otoliths

have been unsuccessful. Do et al. (2006) attempted to validate daily increments of

H. spinosissimus, without success, while Takahashi (2000) presented growth and

0 5

5

0

10

15

20

25

30

35

Nu

mb

er o

f in

crem

ents

10

Time elapsed (days)

15 20 25 30 35

Fig. 2. Relationship between number of days after birth and number of increments for juvenile pipefish Stig-

matopora argus ( ; y = 0·99x− 0·13; r2 = 0·99) and Stigmatopora nigra ( ; y = 0·85x+ 3·3;

r2 = 0·98).

© 2012 The Authors


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Table I. Summary information for Stigmatopora argus and Stigmatopora nigra otoliths detailing the known age, number of increments, total length

range (LT), number of fish (n), mark type [chemical: alizarin complex one (AC) or stress] and estimated ages of juvenile and adult specimens,

respectively. Adult specimens that produced a fluorescent mark were treated with 80 mg l−1of AC. Each row represents a separate event when

specimens were sampled after birth (juveniles) or post-treatment (adults)

Species Juvenile Adult

n

Known age

(days)

Number of

increments

(mean ± s.d.)

LT range

(mm) n

Number of

days held

post-treatment

Number of

increments

(mean ± s.d.) Mark type

LT range

(mm)

Estimated

age (days)

S. nigra 1 10 11 33·10 4 7 6·23 ± 1·27 Chemical 95–100 45–65

1 12 15 34·60 4 7 6·74 ± 0·19 Stress 82–96 70–81

2 20 21 38·00–38·70 2 14 14·00 ± 0·00 Stress 82–91 48–71

1 29 28 47·90

1 30 28 36·50

S. argus 1 0 0 29 1 1 1 Chemical 141 105

1 6 6 38·20 1 7 7 Stress 95 52

18 7 6·75 ± 0·79 25·80–42·50 1 14 14 Stress 139 115

1 26 26 41·00

3 31 30·33 ± 1·61 49·40–52·40

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reproduction data for S. biaculeatus, and was able to rear juveniles up to 63 days

old, but could not observe otolith increments. Hatch marks have been defined in

other seagrass-dwelling species such as the greenback flounder Rhombosolea tapi-

rina Gunther 1862 and the longsnout founder Ammotretis rostratus Gunther 1862

(Jenkins, 1987) and in the scorpion fish Sebastes inermis Cuvier 1829 (Plaza et al.,

2001), but not in syngnathids. While it is acknowledged that a larger sample size

during this research programme would have been desirable, a large number of species

have been validated on smaller sample sizes (Brothers, 1990; Lang & Buxton, 1993).

Unpredicted mortality during captivity and insufficient uptake of marking chemicals

meant that pipefish numbers were reduced and as such results are accepted with

caution.

A large number of studies have shown immersion in AC to be successful in mark-

ing otoliths of fishes (Geffen, 1992; Lang & Buxton, 1993), and in this research

programme, specimens immersed in a concentration of 80 mg l−1 exhibited fluores-

cent marks, while those immersed in 60 mg l−1 did not. While the failure of these

specimens to show a fluorescent mark could be due to an insufficient immersion

concentration of AC, it is not expected to be the result of older or slower growing

fishes failing to incorporate the AC. All specimens used in this study covered a range

of sizes (Table I) that approached the maximum LT for both S. argus and S. nigra

(c. 170 and 160 mm, respectively; K. Parkinson and D. Booth, unpubl. data). Inter-

estingly, though, the syngnathids did exhibit discernible stress marks. Stress marks

are natural structural marks brought about by periods of stress experienced by the

fishes such as temperature change or handling (Brothers, 1990; Geffen, 1992). Dur-

ing this study, marks were observed which corresponded clearly to the handling and

relocation of these pipefishes to aquaria and as such are sufficient to enable analysis

of increments formed during this period (Radtke et al., 1988).

This study shows the suitability of using otoliths as a tool for ageing both juvenile

and adult syngnathids. Daily increments were discernible on both adult and juvenile

S. argus and S. nigra. In addition, a hatch mark was visible on each otolith providing

a point of origin for counting the daily increments.

We would like to thank C. Arnold, A. Wressnig and many others for assistance in thefield. Thanks to the staff at UTS Gore Hill Campus for their help in the aquarium room, N.Richardson, G. Armstrong and S. French, and also the fabulous staff at UTS MAU, M. Phillips,K. McBean, M. Berkhan and especially R. Wuhrer. This is part of RNSH ACEC ethicsapproval number ACEC# RNS/UTS 0711-045A, Animal Ethics approval UTS ACEC 2008-13, NSW Fisheries Scientific Research Permit F94/696(A) and Contribution 63 of SydneyInstitute of Marine Science.

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704 K . L . PA R K I N S O N E T A L .

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© 2012 The Authors


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