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Polar Biol (1995) 15:587-592 © Springer-Verlag 1995 James J. Ruzicka • R.L. Radtke Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy Received: 4 February 1995/Accepted: 17 April 1995 Abstract Larval fish of Antarctica have very narrow rings on their otoliths ( < 1 gm) that may not be re- solved with light microscopy. In this study, age data from the otoliths of larval Nototheniidae (Gobiono- tothen gibberifrons and Lepidonotothen larseni), deter- mined using light and scanning electron microscopy, are compared. Rings 0.4 gm wide were observed on otoliths viewed under electron microscopy; however, light microscopy could only resolve rings >/0.5 gm wide. Scanning electron microscopy is more time con- suming and costly than light microscopy but has greater resolving power and is recommended to vali- date ring counts made using light microscopy in otolith studies with Antarctic larval fish. Introduction Studies of the early life-history strategies of fishes have been facilitated by the development of techniques that provide information about age, growth and develop- ment from otolith microstructure. The first application of otolith techniques to larval fish was by Brothers et al. (1976) who found the daily ring structure in otoliths of larval northern anchovy (Engraulis mordax) that was first noticed in adult fishes by Pannella (1971). Daily ring deposition has since been found to be common among larval fish (Jones 1986). J.J. Ruzicka (~) Department of Oceanography School of Ocean and Earth Sciences and Technology University of Hawaii 1000 Pope Rd. Honolulu, HI 96822, USA R.L. Radtke Hawaii Institute of Geophysics and Planetary Science School of Ocean and Earth Sciences and Technology University of Hawaii 1000 Pope Rd. Honolulu, HI 96822, USA The possibility of daily ring formation in the otoliths of Antarctic fishes was first discussed by Townsend (1980). To date, only four studies have employed otolith microstructure techniques to study the early stages of Antarctic fish. Hourigan and Radtke (1989) validated daily ring formation on otoliths of laboratory-reared Lepidonotothen (Nototheniops) nudifrons. Radtke et al. (1989) generated a growth curve for Trematomus new- nesi larvae using age data from otoliths. Radtke and Kellermann (1991) calculated daily growth rates from the otoliths of larval Pagetopsis macropterus and Pseudochaenichthys georgianus. Kochkin (1986) esti- mated the age of juvenile Champsocephalus gunnari from sub-annual ring counts. In this study, the otoliths of two Antarctic species are examined. Gobionotothen gibberifrons and Lepidonotothen (Nototheniops) larseni are both mem- bers of the family Nototheniidae and the sub-order Notothenioidei. The Notothenioidei is the dominant group of fish in coastal water of the Antarctic. G. gibberifrons, reaching a length of 55 cm, has been harvested commercially since the mid-1970s (Kock 1991). It is found along the northern Antarctic Penin- sula, South Georgia and the Scotia Arc (DeWitt 1971; DeWitt et al. 1990). L. Iarseni, reaching a length of only 24 cm, has a circumpolar distribution throughout the sub-Antarctic islands and the northern Antarctic Peninsula (DeWitt 1971; DeWitt et al. 1990). Antarctic larval fishes pose an additional challenge to the use of otoliths. Their slow growth rates are reflected in very narrow daily rings, often < 1.0 gm (Radtke et al. 1989; Radtke and Kellermann 1991). Such narrow rings approach the resolution limits of conventional light microscopic techniques (Campana et al. 1987). In this study, otoliths are examined with both light and scanning electron microscopy (SEM); and a technique, based upon average ring widths, is developed to estimate the number of rings hidden within unreadable regions. The relative accu- racy of the two viewing techniques is compared.

Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy

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Page 1: Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy

Polar Biol (1995) 15:587-592 © Springer-Verlag 1995

J a m e s J. Ruzicka • R.L. Radtke

Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy

Received: 4 February 1995/Accepted: 17 April 1995

Abstract Larval fish of Antarctica have very narrow rings on their otoliths ( < 1 gm) that may not be re- solved with light microscopy. In this study, age data from the otoliths of larval Nototheniidae (Gobiono- tothen gibberifrons and Lepidonotothen larseni), deter- mined using light and scanning electron microscopy, are compared. Rings 0.4 gm wide were observed on otoliths viewed under electron microscopy; however, light microscopy could only resolve rings >/0.5 gm wide. Scanning electron microscopy is more time con- suming and costly than light microscopy but has greater resolving power and is recommended to vali- date ring counts made using light microscopy in otolith studies with Antarctic larval fish.

Introduction

Studies of the early life-history strategies of fishes have been facilitated by the development of techniques that provide information about age, growth and develop- ment from otolith microstructure. The first application of otolith techniques to larval fish was by Brothers et al. (1976) who found the daily ring structure in otoliths of larval northern anchovy (Engraulis mordax) that was first noticed in adult fishes by Pannella (1971). Daily ring deposition has since been found to be common among larval fish (Jones 1986).

J.J. Ruzicka ( ~ ) Department of Oceanography School of Ocean and Earth Sciences and Technology University of Hawaii 1000 Pope Rd. Honolulu, HI 96822, USA

R.L. Radtke Hawaii Institute of Geophysics and Planetary Science School of Ocean and Earth Sciences and Technology University of Hawaii 1000 Pope Rd. Honolulu, HI 96822, USA

The possibility of daily ring formation in the otoliths of Antarctic fishes was first discussed by Townsend (1980). To date, only four studies have employed otolith microstructure techniques to study the early stages of Antarctic fish. Hourigan and Radtke (1989) validated daily ring formation on otoliths of laboratory-reared Lepidonotothen (Nototheniops) nudifrons. Radtke et al. (1989) generated a growth curve for Trematomus new- nesi larvae using age data from otoliths. Radtke and Kellermann (1991) calculated daily growth rates from the otoliths of larval Pagetopsis macropterus and Pseudochaenichthys georgianus. Kochkin (1986) esti- mated the age of juvenile Champsocephalus gunnari from sub-annual ring counts.

In this study, the otoliths of two Antarctic species are examined. Gobionotothen gibberifrons and Lepidonotothen (Nototheniops) larseni are both mem- bers of the family Nototheniidae and the sub-order Notothenioidei. The Notothenioidei is the dominant group of fish in coastal water of the Antarctic. G. gibberifrons, reaching a length of 55 cm, has been harvested commercially since the mid-1970s (Kock 1991). It is found along the northern Antarctic Penin- sula, South Georgia and the Scotia Arc (DeWitt 1971; DeWitt et al. 1990). L. Iarseni, reaching a length of only 24 cm, has a circumpolar distribution throughout the sub-Antarctic islands and the northern Antarctic Peninsula (DeWitt 1971; DeWitt et al. 1990).

Antarctic larval fishes pose an additional challenge to the use of otoliths. Their slow growth rates are reflected in very narrow daily rings, often < 1.0 gm (Radtke et al. 1989; Radtke and Kellermann 1991). Such narrow rings approach the resolution limits of conventional light microscopic techniques (Campana et al. 1987). In this study, otoliths are examined with both light and scanning electron microscopy (SEM); and a technique, based upon average ring widths, is developed to estimate the number of rings hidden within unreadable regions. The relative accu- racy of the two viewing techniques is compared.

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Materials and methods

Field collection of samples

Fish larvae from the Bransfield Strait were made available from the Research on Antarctic Coastal Ecosystem Rates (RACER) program (Huntley et al. 1991) cruise of the RV Polar Duke (19 January to 2 February 1987). Larvae from South Georgia were collected during two demersal fish surveys of the US Antarctic Marine Living Re- sources (US AMLR). The first was aboard the Polish RV Professor Siedlecki (18 December 1987 to 10 January 1988), and the second was aboard the NOAA ship Surveyor (4 January to 2 February 1989). Both species were collected together on all three cruises. RACER samples were preserved in methyl alcohol, and US AMLR samples were preserved in isopropyl alcohol. A subsample of otoliths was taken randomly from larvae obtained during each 15-, 24-, and 30-day cruise, respectively.

along each otolith was divided into 5-gm or 10-gm sections, between a radius 25 gm from the center to the outer edge. The average ring widths within each consecutive segment were calculated for each subsample (defined by species and cruise) from otoliths with clearly visible rings in that segment. The average ring width within sections with obscured rings was assumed to be the same as that in legible otoliths of the same subsample. The ring number within such sec- tions was easily estimated by interpolation.

The innermost region (3-9 gm wide), between the core edge and the 25-gm radius, contained narrow rings ( < 1 ~tm). In most otoliths this was the most difficult region to resolve clearly. The number of obscured rings within this region was estimated as before but using the average ring widths of the adjacent 25 30-~tm segment.

The estimated number of rings on an otolith is the sum of the visible rings and the number estimated to be hidden within unread- able zones. The estimated age is the average of the three estimated ring counts (or the single estimated ring count when using SEM).

Light microscopy

Sagittae were removed from the fish larvae using very fine dissecting needles and mounted onto glass slides with either "Petropoxy 154" (Palouse Petro Products) or "Crystal Bond" (Aremco Products). "Petropoxy" is permanently set after baking at 300~ for 30 min. "Crystal Bond" may be re-molten to reposition (or remove) otoliths. Otoliths were mounted with the medial (or sulcus) side against the glass and the convex lateral side upward. Large otoliths, opaque to transmitted light, were ground to midplane using either 600 or 1200 grade 3 M sandpaper and polished with either 0.3-or 0.05-gm Bueh- ler Micropolish II aluminum paste on a Buehler polishing wheel.

Otoliths were measured with a video-coordinate digitizer system that included: a compound microscope with a x 10 ocular and x 10, x40, and x 100 (planapo oil immersion) objective lenses, a mono- chrome video camera and monitor, and an H.E. Video Coordinate Digitizer. Measurement resolution was 0.08 ~tm at x 100 magnification.

The following otolith dimensions were measured: longest dia- meter (the tip of the anterior rostrum to the posterior margin), shortest diameter (the dorsal to the ventral margin), shortest radius and the radius of the inner core. The shortest radius was within the excisura ostii (the notch between the antirostrum and the rostrum). The terms for otolith morphology are defined by Hecht (1990).

The distance of each ring from the otolith center was measured (at x 100). Ring measurements were made along the clearest axis. Each

sample was examined with light microscopy on three different days to provide three replicate measurements of all parameters.

Electron microscopy

After being viewed with light microscopy, a randomly selected subset of samples was viewed using a Hitachi S-800 field emission scanning electron microscope. Otoliths were mounted on aluminum stubs with "Petropoxy", medial side down, and ground to midplane. Otoliths were etched with 7% EDTA (ethylenediamine-tetraacetic acid) buffered to a pH of 7.3 with NaOH (for G. 9ibberifrons) or 8% EDTA buffered to a pH of 7.5 with KOH (for L. larseni). Etching times were adjusted for best results (generally 3-5 min for G. 9ibberi- frons and 1-3 rain for L. larseni). Otolith dimensions and the distance of each ring from the center were measured as in light microscopy but only once.

Age estimation

Otoliths contained regions that were unreadable. The number of obscured rings in these regions was estimated. The measured axis

Fig. la, b Gobionotothen 9ibberifrons sagittal otolith, collected off South Georgia (1988/1989) as viewed by a light microscopy and b SEM. Marked features include: 1 rostrum 2 excisura ostii 3 anti- rostrum and 4 inner core edge (or hatch check). The standard length of the larva from which this otolith came was 24.45 mm

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Result~

The standard length distribution, otolith size distribu- tion and age distributions (as derived from both light micro~copy and SEM) of G. gibberifrons and L. larseni are su:mmarized in Table 1. There was no indication of a yolk-sac on any of the larvae.

Exa:3aples of otoliths of each species are given in Figs. 1 and 2, which show imaging by both light microscopy and SlaM of sagittae from the same individuals. The profiles of the ring widths along the measured axis of these two otoliths are shown in Figs. 3 and 4. Figures 3 and 4 also show where rings are estimated to lie withir.L unreadable regions of the otoliths.

The:re are three possible sources of error when estimating the true ring count of an otolith: the reader, the es:imate of the number of obscured rings with- in unreadable regions and the resolution of the micro- scope.

Rea:ler error can be determined by comparing the tl3ree replicate ring counts for each otolith; usu- ally these vary by < 10%. For G. gibberifrons, the range was <_ 5 rings for each subsample. The ranges were ~omewhat greater for L. larseni, _< 6 rings. The difference between observations was > 15% of the estimated age in five L. larseni from the Brans- field S:rait. However, for both species, the largest varia- bility between observations was among the South Georgia 1987/1988 samples where the difference be- tween counts was usually between 10% and 30% of the mean.

The method used to estimate the number of rings contained within an unreadable region is based upon the as.~,umption that the width of those rings is the same as the subsample average (depending upon distance from 1:he otolith's center). The contribution of "esti- mated rings" to the total ring count is substantial in each subsample and is similar whether using light

Table 1 Estimated mean ages (days) using light microscopy and SEM, the mean larval standard lengths (mm) and mean otolith diameters (btm) from each subsample of larvae (each _+ 1 standard deviation). Larvae

Fig. 2a, b Lepidonotothen larseni sagittal otolith, collected off South Georgia (1988/1989) as viewed by a light microscopy and b SEM. Marked features include: 1 rostrum 2 excisura ostii 3 antirostrum and 4 inner core edge (or hatch check). The standard length of the larva from which this otolith came was 20.50 mm

from South Georgia 1987/1988 were collected over a 16-day period, larvae from South Georgia 1988/1989 were collected over 10 days and larvae from the Bransfield Strait 1986/1987 were collected over 11 days

Light microscopy

Age Standard Otolith length diameter a

Electron microscopy

n Age Standard Otolith n length diameter a

Gobionotothen gibber~'ons South Georgia 33 -+ 8 1987/lC,~8

South Georgia 1988/1989 71 ,+ 4

Bransfield St. 1986/1987 38 ,+ 6

Lepidor.)tothen larseni South Georgia 44 ,+ 10 1987/1 c' ~8

South Georgia 69 ,+ 7 1988/19~9

Bransfield St. 1986/1987 39 ,+ 11

12.19 _+ 2.38 126 + 33

22.59 + 1.76 365 _+ 24

16.57 -- 1.98 149 _+ 33

12.25 _+ 1.46 108 _+ 18

18.34 _+ 2.98 213 _+ 26

16.32 +_ 1.57 107 -+ 19

11 40 _+ 12 12.82 + 2.26 132 _+ 34 6

27 78 + 8 23.66 _+ 1.66 395 _+ 42 10

30 41 _+ 12 16.78 _+ 2.91 151 + 30 10

18 58 + 12 13.14 _+ 1.55 126 _+ 24 6

27 68 + 6 18.47 _+ 2.98 201 _+ 20 7

15 57 + 5 17.23 _+ 0.76 119 _+ 6 5

a Longest axis

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590

microscopy or SEM. Among G. 9ibberifrons, estimated rings accounted for 10-15% of the estimated age and among L. larseni they accounted for 15-20%.

The importance of the light microscopy system res- olution limits can be determined by comparing light

microscopy and SEM counts. Sagittae from the same individual were examined using both light microscopy and SEM. A paired Wilcoxon signed ranks test (Hollander and Wolfe 1973) was used to compare the agreement between the two methods (Table 2).

4-

3-

" 0

r -

a

I I 1 I I I I i

A /7/' .-~_ 1 1111 11

O.5 ] ~

O l ' I ' I ' I ' I ' I ' I ' I

5 -

4 -

b71

o o 15 2;

b

1

3; 4o 50 6; 70 Ring Number

8;

Fig. 3a, 5 Profile of the ring widths along the measurement axis of the Gobionotothen gibberifrons otolith shown in Fig. 1 (arrows de- mark estimated rings within unreadable regions); a light microscopy and 5 SEM

"•1.5

~ b~-0.5

0 l , , , , , , , ~ , , , , ,

o 10 20 30 40 so 60 Ring Number

7;

Fig. 4a, 5 Profile of the ring widths along the measurement axis of the Lepidonotothen larseni otolith shown in Fig. 2 (arrows demark estimated rings within unreadable regions); a light microscopy and b SEM

Table 2 Comparison of estimated ring counts in otoliths using light microscopy and SEM. A paired Wilcoxon signed ranks test is used to test the significance of the difference between the two methods for each subsample (Ho: light age = SEM age; H~: light age ~ SEM age)

Mean Mean % Wilcoxon paired sign ranks test difference a difference b T + ~ P n

Gobionotothen 9ibberifrons South Georgia 5 • 5 1987/1988

South Georgia 6 _+ 6 1988/1989

Bransfield St. 2 _+ 7 1986/1987

Lepidonotothen larseni South Georgia 9 _+ 6 1987/1988

South Georgia 0 -t- 6 1988/1989

Bransfield St. 13 _+ 6 1986/1987

10 19.0 0.093 6

8 25.0 0.076 7

2 27.5 0.594 9

15 21.0 0.036 6

- 1 15.0 0.933 7

22 10.0 0.100 5

a SEM ring count-light ring count + 1 standard deviation b (SEM-light)/SEM- 100 ~ Wilcoxon test statistic

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591

Discussi on

The us~ of otoliths to estimate age, growth and devel- opmerLt rates of larval fish requires that ring deposition occurs at a known rate and begins at a known point of development. The periodicity of ring formation has so far be,::n studied in few Antarctic fish but has been shown to be daily in each case. Daily ring deposition has be,~n experimentally validated in three Antarctic species: Trematomus newnesi (Radtke et al. 1989), Lepidonotothen nudifrons adults (Radtke and Hourigan 1990) and larvae (Hourigan and Radtke 1989) and Harpa!tifer antarcticus (White 1991). In addition, Radtk:', and Targett (1984) provided indirect evidence of daily ring deposition in Lepidonotothen larseni. The age when ring deposition begins is not known for either species studied here but is assumed to begin at hatch, formir g the edge of the central core of the otolith. This assumption is supported by the observation that the core edge represents a hatch check in laboratory-reared L. nudifrons (Hourigan and Radtke 1989).

The narrow rings of Antarctic larval fish otoliths increa~,~e the difficulty of retrieving accurate age and growtl: data. When there was a difference between ring counts using the two different microscopy techniques (on the same samples), SEM generally gave the higher estimal:ed ring counts (Table 2). Those subsamples for which Lhere was a great difference were those with the greate:~t variation between the three replicate observa- tions ~.tsing light microscopy (G. 9ibberifrons and L. Iarseni from South Georgia 1987/1988 and L. larseni from tile Bransfield Strait). This suggests that the light microscopy method was deficient in revealing all the rings of the otoliths from these particular subsamples.

The potential usefulness of otoliths can only be real- ized if 1he researcher is able to fully resolve the otolith's microstructure. The theoretical minimum resolution of the light microscope used in this study is given by the equation:

minimlJm resolvable distance = (0.61-2)/NA

where 2 is the wavelength of light used, 0.6 gm for white light, and NA is the numerical aperture of the objective lens, 1.3 in this case (Slayter 1970). Theoretically, the smalle?,t rings that can be resolved are therefore 0.28 gl:a wide. The overall resolution of the entire viewing system (including video camera, digitizer and monitc,r) is probably considerably less. The narrowest rings seen with light microscopy in otoliths from both species were 0.5-0.7 gm wide, but SEM was able to resolve rings 0.4 0.6 gm wide. The light micro- scope ~;ystem used in this study was therefore unable to res(dve rings less than 0.5 t.tm wide. The narrowest rings were located primarily near the center of the otolith

During periods of slow growth, common among pelagic: larvae in cold waters, rings may be deposited

that are narrower than the resolving power of light microscopy (Campana et al. 1987). In cases where rings are less than 1 gm, Campana et al. (1987) recommended using more advanced techniques to enhance resolution.

SEM offers several advantages over light micro- scopy. These include: a resolving power greater than 0.01 gm, a greater depth of field and avoidance of optical artifacts. Unfortunately, SEM requires more involved sample preparation. In this study, each sample viewed with SEM required approximately 2 h of prep- aration. Of those samples so prepared, only 40% had legible microstructures. This, and the cost of electron microscope time (US $30 per hour in this case), pro- hibits large sample numbers. In addition, details of the otolith microstructure may easily become damaged during grinding and EDTA etching during preparation for SEM, thus obscuring some rings. In individual cases, this may be so severe as to offset any gain from greater resolution. Indeed, in this study, the proportion of "estimated rings" (within unreadable zones) to the total ring count was comparable whether using SEM or light microscopy.

In the light of these difficulties with the technique, SEM should not be used to the exclusion of light microscopy in otolith studies on Antarctic larval fish. Light microscopy has been successfully used to age Antarctic larval fish (Radtke et al. 1989; Hourigan and Radtke 1989). However, because of the greater resolv- ing power, the use of SEM is recommended to validate ring counts made using light microscopy in future otolith-based research with Antarctic larval fish.

Acknowledgements We are grateful to David Shafer, Georgia Tien, Mark Huntley, and Valerie Loeb for providing the samples, to Shannon Kayatani for help in the laboratory and to Tina Car- valho and Marilyn Dunlap of the Pacific Biomedical Research Center Electron Microscope Facility. We would also like to thank the scientists and crew of the RV Professor Siedlecki, RV Polar Duke and RV Surveyor. Finally, many thanks are due to Adi Kellermann for his good advice and endless enthusiasm. This study was funded by National Science Foundation grants DPP 85-21017, OCE 84- 15968, DPP-9123017, OCE-9205936, and AMLR. This is School of Ocean and Earth Science and Technology contribution number 3926.

References

Brothers EB, Mathews CP, Lasker R (1976) Daily growth in- crements in otoliths from larval and adult fishes. Fish Biol 74:1 8

Campana SE, Gagn~ JA, Munro J (1987) Otolith microstructure of larval herring (Clupea harengus): image or reality? Can J Fish Aquat Sci 44:1922 1929

DeWitt HH (1971) Coastal and deep-water fishes of the Antarctic. Am Geogr Soc, Antarct Map Folio Ser 15:1-10

DeWitt HH, Heemstra PC, Gon O (1990) Nototheniidae. In: Gon H, Heemstra PC (eds) Fishes of the Southern Ocean. J.L.B. Smith Institute of Ichthyology, Grahamstown, pp 279 331

Hecht T (1990) Otoliths: An introduction to their morphology and use in the identification of Southern Ocean fishes. In: Gon H, Heemstra PC (eds) Fishes of the Southern Ocean. J.L.B. Smith Institute of Ichthyology, Grahamstown, pp 64-69

Page 6: Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy

592

Hollander M, Wolfe DA (1973) Nonparametric statistical methods. Wiley, New York

Hourigan TF, Radtke RL (1989) Reproduction of the Antarctic fish Nototheniops nudifrons. Mar Biol 100:277 283

Huntley M, Karl DM, Niiler P, Holm-Hansen O (1991) Research on Antarctic Coastal Ecosystem Rates (RACER): an interdisciplin- ary field experiment. Deep Sea Res 38:911-941

Jones C (1986) Determining age of larval fish with the otolith increment technique. Fish Biol 84:91 104

Kochkin PN (1986) Analysis of the age sensitive structures and linear growth in the pike glassfish, Champsocephalus gunnari (Channichthyidae). J Ichthyol 25:110 119

Kock K-H (1991) The state of exploited fish stocks in the Southern Ocean a review. Arch Fischereiwiss 41:1-66.

Pannella G (1971) Fish otoliths: daily growth layers and periodical patterns. Science 173:1124 1127

Radtke RL, Hourigan TF (1990) Age and growth of the Antarctic fish Nototheniops nudifrons. Fish Biol 88:557 571

Radtke RL, Kellermann A (1991) Microstructural analysis of growth patterns in the early life history of Antarctic fishes. In: di Prisco G, Maresca B, Tota B (eds) Biology of Antarctic fish. Springer, Berlin Heidelberg New York, pp 101-115

Radtke RL, Targett TE (1984) Structural and chemical rhythmic patterns in the otoliths of the Antarctic fish Notothenia larseni and their application to age determination. Polar Biol 3:203-210

Radtke RL, Targett TE, Kellermann A, Bell J, Hill K (1989) Antarc- tic fish growth: profile of Trematomus newnesi. Mar Ecol Prog Ser 57:103-107

Slayter EM (1970) Optical methods in biology. Wiley-Interscience, New York

Townsend DW (1980) Microstructural growth increments in some Antarctic fish otoliths. Cybium 3e Ser 8:17 22

White MG (1991) Age determination of Antarctic fish. In: di Prisco G, Maresca B, Tota B (eds) Biology of Antarctic fish. Springer, Berlin Heidelberg New York, pp 87-100