An analysis of otoliths from two eels (Anguilla australis and 1

Anguilla dieffenbachii) recovered from a hydroelectric dam 2

turbine in the Tarawera River, New Zealand 3

Paris Achenbach1 4

1. University of Canterbury, Christchurch New Zealand. email: Paris.Achenbach@oberlin.edu 5

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ABSTRACT 7

With the onset of anthropogenic affects on aquatic life including climate change, over-fishing 8

and hydroelectric dam construction, it is more important than ever to understand fish 9

behaviour, life cycles, and migratory journeys. Otoliths provide a wealth of information 10

regarding their biological characteristics; however, they’re also difficult to analyze and 11

interpret, and despite the wealth of existing literature on otoliths, few have provided an all-12

encompassing look at otolith methodologies. The present study experiments on two pairs of 13

eel otoliths, one pair from Anguilla australis and one from Anguilla dieffenbachii, retrieved 14

from the Tarawera River, New Zealand, to examine the biological characteristics of the eels 15

(such as the lifespan and growth rate) using a Scanning Electron Microscope. It also 16

compiles a straightforward and effective approach to methodologies of eel otolith research, 17

from original findings, and from outside recent research. It is important to note that while this 18

paper serves as a review of otolith methodologies, it also presents several new questions 19

regarding the specific otoliths recovered from the Tarawera River. One surprising finding in 20

particular regarding the SEM images was a series of pores in the shortfin eel otolith, 21

suggesting the eel had a disease or other unknown condition. 22

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INTRODUCTION 24

Worldwide freshwater eel (Anguilla species) populations are facing a rapid decline, posing a 25

problem for commercial fisheries, ecosystem health, and cultural diets (Guan et al. 2004; 26

Tesch 2003; Boubee et al. 2008; Chisnall and Hicks 1993; Kelly et al. 2007). Eel is a high 27

commodity for the fishing industry; in 1995, total world eel harvest amounted to about 28

205,000 tons of eel from fishing and aquaculture, which was valued at over US$3.1 billion 29

(Tesch 2003). Further than just being a problem economically, the eel population decline has 30

significant effects for countries that value eels as an important part of their culture, such as 31

China, Japan, and New Zealand (Guan et al. 2004). 32

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In New Zealand, the local Maori people view eels an important part of their diet; not 34

coincidentally, eels naturally contain high amounts of proteins and antioxidants, which have 35

been shown to reduce the risk of heart disease and cancer (Ekanayake et al. 2005). There are 36

two endemic species of eel in New Zealand: the shortfin eel (Anguilla australis) and longfin 37

eel (Anguilla dieffenbachii), the latter of which lives longer and is solely found in New 38

Zealand (Northland Conservancy 2006). 39

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Although it is understood that New Zealand eels are diadromous, meaning they spawn in the 41

ocean but mature in freshwater, little is known about the specifics of where they hatch and 42

travel, and similarly, how they migrate, and what cues they use for navigation. Recent 43

initiatives in discovering eel spawning locations involve satellite tagging, and radio and 44

acoustic telemetry studies (Jellyman 2006, 2009; Jellyman and Tsukamoto 2010; Aarestrup et 45

al. 2009), which have shown that New Zealand eels potentially spawn off the coast of New 46

Caledonia, putting their migration distance over 2,000 kilometers. One satellite in particular 47

showed longfin eels to swim at speeds ranging from 26 to 31 km per day and at depths of up 48

to 980m (Jellyman 2006). 49

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A more common way to examine eel migration and life cycles, however, is by studying their 51

otoliths, which can reveal age and growth rate through back-calculation of annually deposited 52

rings (McCleave 2008; Yokouchi et al. 2011; Tzeng et al. 1993; Marohn et al. 2009; 53

Lecomte-Finiger 1992; Chisnall and Hicks 1993; Graynoth 1999; Jellyman 1995). Further, 54

the otoliths can be used to study the timing of the eel’s entrance into freshwater or saltwater, 55

by analyzing the chemical makeup of the bone (Arai et al. 2003; Tzeng et al. 2005; Tzeng 56

and Tsai 1994; Yokouchi et al. 2011). 57

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There are numerous ways in which otoliths can be prepared and examined, including etching, 59

SEM imaging, staining, burn-and-breaking, sawing, using laser ablation, or a combination of 60

these procedures. This study examines two pairs of otoliths from the Tarawera River using a 61

combination of methods, including with etching and SEM imaging, to compare the 62

differences in ages and size between the two species, as well as understand the potential 63

variables that affect otolith size, morphology, and growth rate. 64

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METHODS 66

Two pairs of eel otoliths were extracted from two eels that had died in a hydroelectric turbine 67

on the Tarawera River, New Zealand. One pair is from a female longfin eel (Anguilla 68

dieffenbachii), which measured to be 160 cm at 8.8 kg. The other pair is from a male shortfin 69

eel, A. australis, which measured to be 100 cm at 1.74 kg. Unfortunately one of the otoliths 70

from the shortfin eel broke in half in transit to the South Island, but it was still possible to 71

examine the two pieces. The otoliths were embedded in resin, then ground to reveal the 72

nucleus and rings, and finally polished on a lap with ½ µm diamond paste (check). They were 73

then examined under a Scanning Electron Microscope four times, under a variety of 74

conditions to obtain the clearest image possible: 75

-With a gold coating 76

-Painted with liquid carbon, drying into a solid conductive surface from the edge of 77

the resin to the edge of the otolith 78

-Etched with Hydrochloric acid for 2-3 minutes 79

After the primary gold covering, the otoliths were grinded again and re-polished, before 80

painting with liquid carbon. 81

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Over the course of four SEM sessions, the otoliths were examined under backscatter and 83

secondary electron microscopy settings. Post-SEM session, images were studied to count the 84

annuli deposits and observe the relationship between the rings, otolith radius, and length and 85

weight of the eel. 86

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RESULTS 88

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The otoliths were difficult to view in the SEM under just a gold coating or painted with liquid 90

carbon; the image was too blurry to see any rings (figure 1a, 1b). However, the etching was 91

highly effective in producing a clear image on the SEM to examine the microstructure. 92

Unfortunately, this meant that the microchemistry of the otolith would not be able to be 93

examined, after altering it with hydrocholoric acid. 94

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Otoliths from Anguilla deiffenbachii 96

The longfin otolith radius measured to be 2.5mm (figure 2) on its longest axis under the 97

SEM, and showed 20 major rings throughout that axis; however, there are clearly many more 98

rings within those rings outlined (figure 3a). On average, major rings occur once every 100 99

µm, but within those 100 µm transects, there are at least 6 or 7 distinct rings (figure 3b). This 100

would put the eel at an age much older than 20. 101

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Otoliths from Anguilla australis 103

The shortfin eel otolith radius measured to be 1.9 mm (figure 4) on its longest axis under the 104

SEM, and showed 22 major rings from the nucleus to the edge, though the rings became 105

increasingly more blurred the further from the nucleus (figure 5a). This otolith too showed 106

multiple rings within rings, suggesting either the eel was older than 22, or that there were 107

false annuli deposits, or perhaps seasonal or daily deposits (figure 5b; figure 6a and 6b). 108

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Pores in Anguilla australis 110

One of the more curious findings of this study was the presence of pores, or perhaps pits, in 111

both of the broken pieces of the Anguilla australis otolith (figure 7a and 7b). Due to lack of 112

time, there are only pictures from one of the pieces, but they were present in both. It’s unclear 113

if they were on opposing sides of the otolith or only on one side, but they clearly 114

conglomerated together in one particular spot on the otolith. 115

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DISCUSSION 117

SEM settings: Backscatter vs Secondary Settings 118

Both the backscatter and secondary settings showed distinct rings after etching; the former 119

showing chemical differences in the otolith, the latter showing topographical differences. 120

Both settings showed rings in the same places (figure 8), indicating that a chemical change is 121

associated with a topographical change in otoliths. 122

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Relationship between Otolith Radius and Eel Length 124

One primary use of studying otoliths is to gauge the relationship between eel length and 125

otolith radius, and find the average rate of growth. Interestingly, the present otoliths fell 126

closely in line with the otolith radius to eel length ratio from a study by Graynoth, which 127

involved 939 longfin and 791 shortfin eels, suggesting that the relationship between the two 128

is curvilinear (figure 9). 129

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Age of Eels 131

Although rough estimates of the eel ages could be found with the images from the SEM, it 132

was difficult to find an accurate or precise age, because the annual bands were often 133

indiscernible from seasonal and daily growth rings. If only the larger bands are counted as 134

annual bands, it would put both eels at an age between 20 and 25 (figures 3a and 5a). 135

However, if the major rings (~6) within the larger rings are counted for the eels, it would put 136

their age at over 100 years old (figure 3b and 5b). 137

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Otolith Pores 139

The otolith pores were clearly not a result of etching, because they did not appear in three of 140

the four otoliths examined which also underwent etching; furthermore, the circles are too 141

rounded to be a result of hydrochloric acid. Unfortunately, because of lack of time in the 142

SEM session, it was unclear whether or not the pits were on opposite sides of the otolith. If 143

they occurred only on one side, then it may be possible they formed around hair follicles 144

during later development, which is what one study found in otoliths from a white sea bass in 145

California (figure 10) (Checkley, unpublished). However, if they formed on both sides, then 146

it must be something else, perhaps a disease or another unknown condition. This is something 147

that should be further investigated. 148

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Problems in Interpretation: Methodologies and Otolith Preparation 150

Otolith interpretations can be easily affected by the process in which they are prepared and 151

studied. For example, depending on how you orient the otolith in resin and how far down you 152

grind and polish the otolith, you can get an inaccurate ring count, and thus age, of the otolith 153

(figure 11) (Panfili and Ximenes 1992). Similarly, eels in colder water grow slower than in 154

warm water, making rings closer together and difficult to discern from one another; this is 155

particularly a problem in glacial lakes of New Zealand, where eels are only growing about 156

9mm/year (especially difficult for really old eels, up to 106 years) (Jellyman 1995). Further, 157

the sampling techniques of otolith trace elements such as diameter of sampling spot could 158

affect the concentration of elements and reconstruction of migratory history of fishes 159

(Yokouchi et al 2011). Lastly, the otolith radius is measured on its longest axis in most 160

studies, but the longest axis depends on how you orient and grind them (figure 12) (Graynoth 161

1999). 162

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In this particular study, it was difficult to find an accurate age count under the SEM for a 164

number of reasons, including: a) the charge buildup that occurs and causes black spots in 165

imaging after time, thus the examiner must operate with maximum efficiency to find the best 166

images as early as possible; b) photos later can be difficult to interpret; c) it’s difficult to get 167

an overall picture of larger otoliths (ie >2mm radius on its long axis) because of the narrow 168

scope of the SEM lens. 169

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Problems in Interpretation: Complications in Ontogenic Development 171

In addition to preparation issues causing a conflict in otolith interpretation, there have been 172

numerous studies showing other biological variables affect otolith morphology and growth 173

rings. 174

1. Otolith rings are often thicker in one section than another, and often overlap or 175

coalesce as well (figure 3b, figure 12). Thus, it’s difficult to get an accurate length/age 176

to otolith radius ratio, ie a growth rate to otolith radius ratio, because the otolith radius 177

is inconsistent. 178

2. Bands, or checks, are formed by changes in calcium and protein deposition rates 179

due to sudden changes in temperature, and possibly other factors, as opposed to 180

simply annual deposits (Graynoth 1999, Jellyman 1995). Similarly, irregular variation 181

in width of summer bands could represent migrations between different habitats, 182

because rate of maturation is affected by temperature, pressure and swimming 183

(Jellyman and Tsukamoto 2010). Thus, rings could potentially be due to starvation, an 184

increase or decrease in swimming, or other biogenic or environmental variables, as 185

opposed to a temporal variable. 186

3. There’s a delay in the incorporation of different Sr:Ca ratios depending on ambient 187

water – time lag of at least 10 days and up to 30-60 days in Anguilla japonica; and 188

further, strontium incorporation can also be affected by biological factors like 189

temperature and growth (Yokouchi et al. 2011), both of which make it difficult to 190

assess an accurate timeline for eel migration using strontium-calcium ratios found 191

through chemical analysis of the otolith 192

4. The area of the otoliths can increase when carbon dioxide levels in the ocean water 193

increases due to acidification – an affect that would take place during the leptocephali 194

phase, and could potentially increase the area of the otolith by a significant percent, 195

thus throwing off the otolith radius to length/age ratio (Checkley et al 2009) 196

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CONCLUSION 198

Many questions remain regarding spawning locations of both eel species, but particularly of 199

A. deiffenbachii, of which only one eel has been observed to travel to New Caledonia, and no 200

larvae have ever been found in the ocean (Kuroki et al. 2008; Jellyman and Tsukamoto 201

2010). Due to the rapid decline in their population numbers, it is imperative to understand eel 202

behaviour and migration if we want to set up conservation schemes and protect them from 203

potential obstacles to their migration, such as hydroelectric dams. 204

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There has been a multitude of research published on eel otolith growth, microstructure, and 206

microchemistry, and yet the data is quite convoluted and makes it difficult to come up with 207

many accurate conclusions. Otolith microstructure studies provide an important base for our 208

understanding of eel migration and life cycles, but when it comes to preserving their 209

population, a more practical approach is necessary. As of now, satellite tagging and acoustic 210

and telemetry studies provide the best option for establishing effective protection measures, 211

as they can tell us a) the origin of eel spawning, b) their migratory paths (distance, location 212

etc), c) time taken during migration, d) eel movement and behaviour within the water 213

column, e) movement within lakes, and f) how much of their migration is due to using 214

oceanic currents for travelling efficiency, versus how much is due to actual navigation and 215

swimming. From there, it may even be possible to understand the environmental cues and 216

conditions that promote or guide their migration paths, whether they are lunar 217

phases/declination, water temperature, or magnetic navigation. Once we understand how and 218

why they are migrating, it may be possible to establish how anthropogenic affects such as 219

global climate change and hydroelectric dam construction may impact their migration and 220

ultimately survival. 221

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ACKNOWLEDGEMENTS 223

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I’d like to thank Kerry Swanson for the endless help with the four SEM sessions and otolith 225

preparation; Travis Horton and Nan Hauser for promoting my interest in animal migration; 226

Bill Kerrison and Mick Kearson for the otoliths and inspiration; Rob Spiers for the diamond 227

paste, sandpaper and entertaining stories; Don Jellyman at NIWA for taking time out of his 228

busy day to explain eel migration and otoliths; and Josh Blackstock for helping me all along 229

the way. 230

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REFERENCES 235

Arai T, Kotake A, Lokman PM, Tsukamoto K (2003) Migratory history and habitat use by 236 New Zealand freshwater eels Anguilla dieffenbachia and A. australis, as revealed by otolith 237 microchemistry. Ichthyological Resesarch 50: 190-194 238

Boubee J, Jellyman D, Sinclair C (2008) Eel protection measures within the Manapouri 239 hydroelectric power scheme, South Island, New Zealand. Hydrobiologia 609:71-82 240

Checkley DM, Dickson AG, Takahashi M, Radich A, Eisenkolb N, Asch R (2009) Elevated 241 CO2 Enhances Otolith Growth in Young Fish. Science 342:1683. 242

Chisnall BL, Hicks BJ (1993) Age and growth of longfinned eels (Anguilla dieffenbachii) in 243 pastoral and forested streams in the Waikato River basin, and in two hydro-electric lakes in 244 the North Island, New Zealand. New Zealand Journal of Marine and Freshwater Research 27: 245 317-332 246

Ekanayake PM, Park GT, Lee YD, Kim SJ, Jeong SC, Lee J (2005) Antioxidant Potential of 247 Eel (Anguilla Japonica and Conger Myriaster) Flesh and Skin. Journal of Food Lipids 12:34-248 47 249 250 Graynoth, E (1999) Improved otolith preparation, ageing and back-calculation techniques for 251 New Zealand freshwater eels. Fisheries Research 42: 137-146 252 253 Guan R, Xuehong W, Guozhen K (1994) Age and growth of eels Anguilla japonica in a 254 Chinese river. Journal of Fish Biology 45: 653-660. 255

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Jellyman D (2009) A review of radio and acoustic telemetry studies of freshwater fish in New 260 Zealand. Marine and Freshwater Research 60: 321-327 261

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Kuroki M, Aoyama J, Miller MJ, Watanabe S, Shinoda A, Jellyman DJ, Feunteun E, 267 Tsukamoto K (2008) Distribution and early life-history characteristics of anguillid 268 leptocephali in the western South Pacific. Marine and Freshwater Research 59: 1035-1047 269

Lecomte-Finiger R (1992) The crystalline ultrastructure of otoliths of the eel (A anguilla L. 270 1758). Journal of Fish Biology 40:181-190 271

Marohn L, Prigge E, Zumholz K, Klugel A, Anders H, Hanel R (2009) Dietary effects on 272 multi-element composition of European eel (Anguilla anguilla) otoliths. Mar Biol 156:927-273 933 274

McCleave JD (2008) Contrasts between spawning times of Anguilla species estimated from 275 larval sampling at sea and from otolith analysis of recruiting glass eels. Mar Biol 155:249-276 262 277

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Figure 3a (top) and 3b (below): An overlook of the longfin otolith. The short, dark transects mark the major annual deposits, but smaller bands in between could represent annual rings as well. The opaque zone on the side could possibly be a result of incorrectly orienting the otolith in the resin. A close-up of rings on the longfin otolith. Major bands were outlined in black, totalling seven on the left. Note the crack running through the center, causing a discrepancy in the rings between the left and the right. This was probably biological in origin, as opposed to a result of the otolith preparation

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Figure 5a (top) and 5b (below): An overlook of the shortfin otolith. Darker transect lines mark the annual deposits, 22 in total, though some of the lines are more ambiguous than others. The annual lines start at the first major dark deposit, which is understood to be when the eel migrates into freshwater, at around 10-11 months old. Within the approximate 10-20 micron-wide rings, there are anywhere from 5-10 more rings inside.

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Figure 1a and 1b: An SEM photograph of the longfin otolith with a carbon coat, and without hydrochloric acid etching. Note some grinding and dust marks.

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Figure 2. Burnt otolith from a 17-year-old longfin eel caught in the Te Maari Stream, courtesy of Graynoth 1999. Note the coalesced annuli (middle left).

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Figure 8. Potential daily or seasonal deposits in the shortfin eel, each about a micron in width, under both backscatter and secondary settings (backscatter showing chemical differences, secondary showing topographical differences). Note that in both settings, rings appear in the same place, indicating that chemical and physical differences in the otolith are linked.

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Figure 6a (top) and 6b (bottom). Rings within annually deposited rings in the shortfin eel.

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Figure 4. Otolith radius of the shortfin eel. Radius was taken on the longest axis.

Figure 2. Otolith radius of the longfin eel.

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Figure 9. Eel length versus otolith radius for both species, courtesy of Graynoth 1999. The shortfin eel length:otolith radius ratio fall closely in line with the other samples. The predicted and actual values for the longfin eel went off the graph, but also seem to follow the general trend.

Figure 10. A picture of a white sea bass otolith containing pits on one side, believed to be a result of the otolith growing around the hair follicles. Courtesy of David M. Checkley, 2009.

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Figure 7a (top) and 7b (below). Pores in the shortfin eel otolith. They appeared in one large cluster, with a few scattered around the outside edge as well. Pores range from approximately 5-20 microns in diameter.

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Figure 11. Diagram of a theoretical otolith with growth rings and the effect of different grinding planes, courtesy of Panfili and Ximenes (1992). Note that, depending on the grinding plane, the otolith radius and length between bands changes, as does the number of rings available to count if oriented incorrectly.