Age and growth of the Antarctic lanternfish, Electrona antarctica (family myctophidae), based on microstructural analysis of sagittal otoliths

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Age and growth of the Antarctic lanternfish, Electrona antarctica (family myctophidae), based on microstructural analysis of sagittal otoliths

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Title:
Age and growth of the Antarctic lanternfish, Electrona antarctica (family myctophidae), based on microstructural analysis of sagittal otoliths
Creator:
Greely, Teresa Marie
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Tampa, Florida
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University of South Florida
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English
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viii, 115 leaves : ill. ; 29 cm.

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Lanternfishes -- Growth -- Antarctic ocean ( lcsh )
Otoliths ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF ( FTS )

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Thesis (M.S.)--University of South Florida, 1994. Includes bibliographical references (leaves 100-106).

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University of South Florida
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Universtity of South Florida
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All applicable rights reserved by the source institution and holding location.
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030026096 ( ALEPH )
31235260 ( OCLC )
F51-00109 ( USFLDC DOI )
f51.109 ( USFLDC Handle )

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AGE AND GROWTH OF THE ANTARCTIC LANTERNFISH, ELECTRONA ANTARCTICA (FAMILY MYCTOPHIDAE), BASED ON MICROSTRUCTURAL ANALYSIS OF SAGITTAL OTOLITHS by TERESA MARIE GREELY A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science University of South Florida May 1994 Major Professor: Dr. Joseph J. Torres, Ph.D.

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Graduate School University of south Florida Tampa, Florida CERTIFICATE OF APPROVAL Master's Thesis This is to certify that the Master's Thesis of TERESA MARIE GREELY with a major in Marine Science has been approved by the Examining committee on March 25, 1994 as satisfactory for the thesis requirement for the Master's Science degree Examining committee: Jlflhp V. Jr_., .Ph.D. Member: Thomas L. Hopkins, Ph.D.

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DEDICATION thesis is dedicated to my grandparents, orval and Betti Root.

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ACKNOWLEDGMENTS Numerous individuals were instrumental in the success of this acacemic endeavor. I would like to thank my colleagues and friends for their unceasing support, assistance, and I extend my gratitude to my major professor, Dr. Jose rorres for the inspiration and support for this research. I thank my committee members, Dr.'s Jack Gartner and Thomas Hopkins. I thank Dr. Gartner for his many critical reviews of the manuscript and continuous assistance in reading and interpreting the otolith morphologies. I wish to extend my thanks to the Florida Department of Environrr:ent.al Protection (DEP) for the use of equipment and staff durin9 thls research. A special thanks to Earnest Truby, Iliana Quintero-Hunter, Michael Murphy, Kevin Peters, and Renee Bishop. My gratitude to the students, staff and faculty o the Department of Marine Science. Special thanks are extende: d to Tony Greco, Greg Tolley, Eric Wright, and Mark Peebles their technical assistance and support as friends and And finally, I wish to thank my friends for their constant encouragement and for ensuring I take care o f myself. I'hanks to Hepsi Zsoldos, Catherine Saenz, Greg Tolley David MuL: 5.ns, Patricia Hernandez, and Mom and Dad.

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TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT vi INTRODUCTION 1 BACKGROUND 11 General:AgeandGrowth ........................... 11 OtolithMicrostructure: DailyGrowth ............. 14 Age and Daily Growth of Myctophids ............... 15 METHODS 19 Errlbedd ing ................. ................... 2 2 Grinding and Polishing ....................... 23 Etching ................ ................................ 2 7 Acetate Replication and Scanning Electron Microscopy . 29 Data Analysis .............. ......................... 31 RESULTS 33 General ................................................ 33 Methodological Results ................................ 37 Otolith Morphology: External ......... ... ........ 38 Otolith Morphology: Internal .................. 41 Growth Rates and Life Spans .................... 67 DISCUSSION 76 Previous Studies ...................................... 77 Validation ............................................. 80 Sagitta Morphology ................................ 81 Physiology /Bioenergetics ........................ 85 Ecology ................................................ 9 0 LITERATURE CITED 100 i

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APPENDICES APPENDIX 1. APPENDIX 2. APPENDIX 3. 107 EXPANDEDMETHODOLOGY .... ............... 108 INCREMENT COUNTS WITHIN THE LARVAL ZONE ( LZ) .............................. 110 INCREMENT COUNTS WITHIN THE POSTMETAMORPHIC ZONE (PMZ) ................. 114 ii

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LIST OF TABLES TABLE 1. Spring (1983) and Fall (1986) trawl data from AMERIE Z cruises during which otoliths were collected ........................................ 3 4 TABLE 2 Morphometric data recorded from sagittae of Electrona antarctica .............................. 42 TABLE 3 Results of Tukey's multiple range test of means for standard length, total radius and larval zone radius by sex for Electrona antarctica ...... 43 TABLE 4. Linear regression equations of standard length (Length in mm) on sagitta total radius (Radius in mm) for Electrona antarctica .......... .. .. ..... 43 TABLE 5 Equations for the overall growth curve and individual growth curves for ma l e and female Electron a antarctica ...................... ........ 75 TABLE 6 Maximum age estimates for 6 tropicalsubtropical and 2 subantarctic myctophid species based on primary growt h i ncrements ........ 95 iii

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LIST OF FIGURES FIGURE 1 The zoogeographic distribution of the endemic myctophid, Electrona antarctica, inhabiting the Southern Ocean (adapted from McGinnis 1982) ... 2 FIGURE 2. Electrona antarctica, the endemic lanternfish of the Southern Ocean ............................. 5 FIGURE 3. Diel vertical distribution of Electrona antarctica collected during the Spring (1983) and Fall (1986) AMERIEZ cruises (adapted from Lancraft et al., 1989) ............................ ? FIGURE 4 Sampling areas in the Weddell Sea (A) and the Scotia Sea (B) .................................. 21 FIGURE 5. Lateral view of Electrona antarctica sagittal otolith, showing the radial axes measured for morphometric analyses and directional axes ....... 24 FIGURE 6. Pooled (1983 and 1986) length distribution of individuals from which sagittae were extracted ... 35 FIGURE 7. External morphology of sagittae of immature, male, and female Electrona antarctica from small, intermediate and large size ranges ........ 39 FIGURE 8. Linear regression of standard length on sagitta total radius for pooled data of all individuals, n=117 ............................... 44 FIGURE 9. Linear regression of standard length on sagitta total radius for unpooled data arrayed by gender ................................ 46 FIGURE 10. Overall view of internal morphology of sagitta from Electrona antarctica ( 82mm, female) ......... 49 FIGURE 11. Entire larval zone with metamorphic check and accessory primordia visible .................. 52 FIGURE 12. "Daily" and subdaily increments within the larval zone of Electrona antarctica ............. 54 iv

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FIGURE 13. All increment counts in LZ (n=60) regressed against standard length of indi victuals ........... 57 FIGURE 14. Low and high magnification SEM views of an etched frontal section showing the threedimensional relief pattern characterizing samples ............................... ........... 61 FIGURE 15. SEM photomontage of the PMZ illustrating the daily increments within this zone ................ 63 FIGURE 16. Low magnification SEM view of an etched frontal section showing the internal structures medially towards the periphery ........ 65 FIGURE 17. High magnification SEM view showing the growth increments juxtaposed to two "monthly" time marks ................................. ...... 68 FIGURE 18. Linear model of growth from pooled data for all Electrona antarctica ......................... 70 FIGURE 19. Growth rates estimated for male and female Electrona antarctica ............................. 73 FIGURE 20. Backcalculated birthdates estimated for the 32 indi victuals used for age and growth analysis ..... 83 FIGURE 21. Growth rate estimates for antarctic and subantarctic myctophids .......................... 9 3 FIGURE 22. Growth rate estimates for cold temperate and polar species ................................... 97 v

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AGE AND GROWTH OF THE ANTARCTIC LANTERNFISH, ELECTRONA ANTARCTICA (FAMILY MYCTOPHIDAE), BASED ON MICROSTRUCTURAL ANALYSIS OF SAGITTAL OTOLITHS by TERESA MARIE GREELY An Abstract A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science university of South Florida May 1994 Major Professor: Dr. Joseph J. Torres, Ph.D. vi

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Throughout the oceanic pelagial of the Southern Ocean, the lanternfish, Electrona antarctica, is the numerically dominant fish. Electrona antarctica is important to the pelagic ecosystem as a major component of the fish biomass, a dominant krill predator, an important prey item of seabirds, and a pivotal species in energy transfer from macrozooplankton to higher trophic levels. The focus of the present study was to resolve primary growth increments from sagittal otoliths to estimate the average life span and calculate growth rates for antarctica. Sagittae were prepared for analysis using three sequential techniques: thin-section grinding and polishing, etching, and scanning electron microscopy (SEM). Sagittae were examined from immature, male and female individuals encompassing the postmetamorphic size range from 23-103 mm SL Montages of SEM photomicrographs were constructed to enumerate microincrements along the entire growth axis. Based on increment width (0. 8 1 2 microns} and continuity, these microincrements were assumed to be deposited on a daily basis. Results suggest a larval stage of 30-47 days and a maximum life span of 3 1/2 years. Growth patterns between males and females were similar, but the growth rates were significantly different for each. 0.072 mmjday for females and 0.05 Growth rates averaged mmjday for males. Information on the age and growth rates of antarctica ?rovide important data addressing the ecology and population iynamics of the pelagic Antarctic ecosystem. vii

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Abstract Approved: Major Joseph J. Torres, Ph.D. Professor, Department of Marine Science Date Approved: viii

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1 INTRODUCTION The fish fauna within the Southern Ocean is characterized by a low diversity of species and a high level of endemism. The most abundant oceanic mesopelagic fishes in Antarctic waters are the lanternfishes (family Myctophidae), deepsea smelts (family Bathylagidae), barracudinas (family Paralepididae), and bristlemouths (family Gonostomatidae; Andriashev, 1965; Hempel et al., 1983; Kock, 1987). Taxa from these families account for greater than 95% of the biomass of mesopelagic fishes Weddell-Scotia sea region in the upper 1000m of the (Lancraft et al., 1989). Representatives of the family Myctophidae will be the focus of the present study. Among the myctophids of the Southern Ocean, three species of the genus Electrona are abundant, antarctica {GUnther, 1878), carlsbergi {Taning, 1932), rissoi {Cocco, 1829). McGinnis {1977) has shown a strong correlation between the distributional patterns of Electrona and the hydrography of the Southern Ocean (Figure 1). Electrona is the only myctophid genus found in both warm water and the Southern Ocean (McGinnis, 1982). Electrona antarctica is considered a polar species, exhibiting an Antarctic pattern of distribution (Hulley, 1981). Electrona carlsbergi is characterized by an

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Figure 1. The zoogeographic distribution of the endemic myctophid, Electrona antarctica, inhabiting the Southern Ocean (adapted from McGinnis 1982). The region of the Polar Front and subtropical convergence are delimited by the southern and northern shaded areas.

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3

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4 Antarctic Polar Front pattern, being restricted to this region as adults, whereas Electrona rissoi inhabits warm waters, with the southern limits occurring around the Subtropical Convergence. Of the three species, antarctica is the numerically dominant myctophid in midwater trawl samples taken throughout the Southern Ocean region (Rowedder, 1979b; Hulley, 1981; 1989) McGinnis, 1982; Linkowski, 1987; Lancraft et al., Electrona antarctica is also the only lanternfish endemic to the Southern Ocean as it is restricted to waters south of the Antarctic convergence. Morphologically, in comparison to most temperate and tropical species of lanternfish, antarctica is unusually large (82-110mm SL); it is deep bodied anteriorly with a prominent head region followed by a tapering caudal region (Figure 2). Males and females exhibit sexual dimorphism both in size (males smaller) and the presence of luminescent scales on the caudal peduncle (dorsal in males and ventral in females). A shallow living myctophid, Electrona antarctica is a strong diurnal vertical migrator occupying a nighttime range of 0-710m and a daytime range of 170-980m with peak abundances occurring at 0-300m at night and 650-920m during the day (Figure 3). The diet of postmetamorphic antarctica (30-110mm SL) consists primarily of euphausiids, copepods, and ostracods (Hopkins and Torres, 1989; Lancraft et al., 1991). Electrona antarctica virtually always dominates in numerical

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Figure 2. Electrona antarctica, the endemic lanternfish of t.hP. southern Ocean. Female, 90mm SL.

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6

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Figure 3. Diel vertical distribution of Electrona antarctica collected during the Spring (1983} and Fall (1986) AMERIEZ cruises (adapted from Lancraft et al., 1989}. n = numbers of individuals collected.

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0 200 -s 400 I 1a.. w 600 0 800 1983 1986 DAY NIGHT DAY NIGHT t ...... ] =:= : =:=:= :=:=:=:=:=:=:=:=:=:=:=:::=:=:=:=:::=:=:=:=:I r----1 r----l-L----n=109 1 000 t I I I I I I 8 6 4 2 0 2 4 101 3 5 7 9 INDIVIDUALS/104m3 00

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9 abundance (0.25 individualsjm2 ) and biomass (388-405 mgDWjm2)in samples collected throughout the Weddell and Scotia sea regions (Torres and Somero, 1988b; Hopkins and Torres, 1989; Lancraft et al., 1991). Clearly, E antarctica is an ubiquitous component of the Antarctic mesopelagic ecosystem. Currently, information regarding Electrona antarctica includes distribution and zoogeography, (Andriashev, 1965; Voronina, 1973; McGinnis, 1977, 1982; Hulley, 1981), proximate composition (Donnelly et al., 1990), metabolism (Torres and Somero, 1988a, b), trophodynamics (Rowedder, 1979b; Lancraft et al., 1991) and reproduction (Rowedder, 1979a). There is a notable lack of information available on the growth rates and life spans of E antarctica and for Antarctic mesopelagic fishes in general (Linkowski, 1987), particularly when compared to the information on tropical, temperate, and subarctic species. Accurate age determinations provide basic life-history information and are imperativ e for describing a species population dynamics. Two age-related studies have been attempted for E antarctica (Row edder, 1979a; Linkowski, 1987), but yielded inconclusive results, further emphasizing the clear need for this fundamental information. The lanternfish, Electrona antarctica, is significant to the pelagic ecosystem as a major component of the fish biomass and as a dominant krill predator (Williams, 1985; Kock, 1987; Lancraft et al., 1989). A s an important prey item of seabirds in open waters of the Antarctic region (Ainley et al., 1986),

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10 Electrona antarctica plays a pivotal role in the transfer of energy from the macrozooplankton (e.g. krill, Euphausia superba) to higher trophic levels (e.g. Antarctic seabirds and mammals) The focus of the present study was to describe the external and internal morphology of the sagittal otoliths of the numerically dominant Antarctic lanternfish, Electrona antarctica, collected from the Weddell-Scotia sea region using microstructural techniques to resolve primary growth increments, to determine the average life span, and to calculate growth rates. These data will allow better evaluation of the role of antarctica in the Southern Ocean ecosystem.

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11 BACKGROUND General: Age and Growth Age determinations of fishes provide basic life history information including age at spawning, maturity, and mortality and population age structure. Information on growth rates of Antarctic fishes is necessary to formulate population dynamics and production models, and to allow comparisons with the growth patterns of fishes in temperate, tropical, and subarctic waters (Campana and Neilson, 1985). In order to address one or all of these topics, an accurate and reliable method of ageing fishes is imperative. Conventional methods of ageing utilize a variety of bony structures, e.g. scales, otoliths, opercular bones, fin rays and vertebrae. The underlying assumptions of interpreting age from bony structures are that periodic features are formed at a constant frequency, and that the distance between consecutive features (e.g. annual bands) is proportional to fish growth. The use of otoliths is accepted as the most accurate method for age determination in most fish species (Six and Horton, 1977; Panella, 1980; Campana and Neilson, 1985). otoliths offer several advantages over other hard structures. Unlike scales, they are not susceptible to

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12 resorption (Mugiya and Watabe, 1977; Marshall and Parker, 1982; Geen et al., 1985). otoliths undergo little alteration once formed (Campana, 1983) and are often the first calcified structure to appear during early development. In addition, otoliths can be utilized when scales are absent or are delicate and easily lost during capture of the fish, as generally occurs with the myctophids. Popper and Coombs (1980) have suggested that in fishes, otoliths function in hearing, balance, and detection of linear acceleration. Otoliths are structures located in the membranous labyrinth of the inner ear and are composed mainly of calcium carbonate in the form of aragonite (Degens et al., 1969) Three pairs of otolith are formed in teleosts; the sagittae, lapilli and asterisci. The sagittae are most commonly used for ageing studies because they are the largest and most easily removable. However, recent studies have suggested differential growth of the otoliths resulting in variable ages for the same individual. The otolith grows in primarily a radial plane as a fish increases in length. The fact that otolith size closely correlates with fish size underscores its utility for growth studies (Campana and Neilson, 1985). Growth is incremental within the otolith, i.e. accretional processes are governed by generally fixed temporal controls. Age determinations using otoliths are made by counting growth increments which appear as concentric rings. A growth increment is a bipartite

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13 structure consisting of incremental (calcium carbonate) and discontinuous (protein) zones. The discontinuous zone is a zone of decreased aragonite deposition so that an underlying proteinaceous matrix, called otolin, becomes apparent. Incremental otolith growth occurs through differential deposition of calcium carbonate and protein. Visible increments may be subdaily, daily, lunar, seasonal (annual), or growth discontinuities (e.g. recording an unusual event in rhythmic growth) Most ageing studies on high latitude fish species are based on ages determined from annual rhythmic depositions (seasonal "rings") in otoliths as time marks (Bagenal, 1974; Blacker, 1974; Williams & Bedford, 1974). The otoliths of Antarctic fishes do not appear to contain interpretable annual deposition patterns, perhaps due to the lack of distinct periodicity in Antarctic hydrographic conditions (Radtke, 1984) Similar complications have been documented for tropical species where seasonally induced marks are irregular, indistinct or absent. Otoliths from Antarctic fish are usually small and difficult to analyze using conventional methods. However, finer scale observations of these otoliths hold promise for making accurate age determinations, as microstructural techniques have been successfully applied to several temperate, subtropical, and tropical species.

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14 Otolith Microstructure: Daily Growth Pannella ( 1971) first described microstructural increments in sagittal otoliths which were found to have been deposited on a daily basis. The incremental growth occurs through differential deposition of calcium carbonate (aragonite) and protein (otolin) over a 24 hour period. Daily growth increments have since been found in the otoliths of several temperate and tropical species from larval to adult sizes (Pannella, 1974, 1980; Brothers et al., 1976; Brothers, 1979; Haake et al., 1982; Campana and Neilson, 1985; Prince et al., 1991), establishing the reliability of otolith microstructure analyses. Application of microstructural techniques to otoliths of Antarctic fishes has proven successful for the few species analyzed. The focus has been almost exclusively on the age and growth of the dominant coastal dwelling, demersal species of the suborder, Notothenioidei (Wohlschlag, 1961; North et al., 1980; Townsend, 1980; Radtke, 1984; Radtke and Targett, 1984; Radtke and Hourigan, 1990). These taxa are abundant shallow water dwellers that can be maintained in captivity for validation experiments. Townsend (1980) first described microstructural growth increments in five coastal, shallow water Antarctic fish (Aethotaxis mitopteryx, Champsocephalus gunnari, Notothenia gibberifrons, N larseni, and N rossii) utilizing SEM

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techniques. Radtke and Targett (1984) 15 also resolved microstructural increments in the sagittal otolith of N larseni by SEM analysis. Radtke and Hourigan (1990) studied the age and growth of N. nudifrons using SEM analysis of microincrements. They also conducted validation experiments for the ageing technique by tetracycline incorporation into the otoliths of individual fish. This was the first study to confirm that the microincrements found within the otoliths of Antarctic fishes are deposited on a daily basis. Age and Daily Growth of Myctophids Although the abundance and importance of myctophids have been well documented, age and growth studies based on microstructural increments in otoliths are limited. Gj0saeter (1987) examined primary growth increments from the otoliths of six tropical myctophid species. H e demonstrated the presence of primary growth increments using light microscopy, acetate replication, and SEM. Methot (1981) used primary growth increments to age the myctophid, Stenobrachius leucopsarus, from California waters. Young et al. (1988) utilized primary growth increments to estimate age and growth of Lampanyctodes hectoris from continental slope waters of eastern Tasmania. Each of the forme r studies assumed a daily period for the growth marks observed, with no direct verification. Gartner (1991) successfully enumerated and validated daily deposition

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16 of microincrements for three myctophid species, from the Gulf of Mexico. This study offered the first direct verification of daily increments in otoliths of lanternfishes by use of marginal increment analysis. Few papers have addressed age and growth of Antarctic myctophids (Rowedder, 1979a; Linkowski, 1985, 1987; Zasel'sliy et al., 1985), although their abundance and contribution to the Antarctic ecosystem has been well established. No study to date has been successful in using otolith microstructural techniques for age determinations of Electrona antarctica. The first published age data for antarctica was contributed by Rowedder ( 1979a} Rowedder found that age determination wa s not possible using the otoliths or opercula of this species. Based on length frequency distributions, he concluded that antarctica attains an age of 3 years. Three distinct peaks in size groups were evident for both females and males, presumably indicating successive age groups. The length frequency data also suggested sexual dimorphism, in which females (maximum s tandard length, 104mm) were noticeably larger than males (maximum standard length, 87mm). Rowedder's conclusions were viewed cautiously, as several inaccuracies may be associated with length frequency analysis for age and growth determinations. This is problematic because older fishes asymptotically approach a maximum size, forming an allinclusive mode at the largest size class.

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17 Linkowski (1987), using length frequency analysis and otoliths, determined ages and growth rates of four Electrona species; ventralis. antarctica, carlsbergi, rissoi, The age determined for antarctica using otolith zonal macrostructure was nearly four-fold that reported by Rowedder (1979a), based on length frequency analysis. Linkowski's length frequency distributions showed different modes for males and females, as suggested by Rowedder' s results, but no distinct size groups were apparent. Despite attempts with both light and scanning electron microscopy, he was unsuccessful in finding primary growth increments in the otoliths of this species. Therefore he made direct counts of zonal macrostructures, alternating hyaline and opaque zones, assuming that one opaque and a consecutive hyaline ring represented an annual time mark. Analysis with this technique concluded that males reached an age of 8 years and females up to 11 years. Fine structure analysis was attempted by Linkowski (1987) for Electrona carlsbergi using light and SEM, but the primary rings were not clear and results were inconclusive. The zonal structure of carlsbergi otoliths was enumerated in a similar manner to antarctica otoliths, resulting in a maximum age of 5.5 years. For both species the zonal patterns were assumed to represent annual deposition. Linkowski successfully resolved primary growth increments from the otolith of Electrona rissoi and (Metelectrona)

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18 ventralis using acetate replication. Each primary ring was assumed to be daily and ages were determined from direct counts. Electrona rissoi attained an age of 1.5 years, while ventralis reached 2 years. Linkowski suggested that the growth parameters estimated for the four species of Electrona conformed well with their patterns of distribution. He further concluded a slower growth rate for species occupying colder Antarctic waters throughout their life cycle (e.g. antarctica) while those which migrated to Antarctic waters as adults carlsbergi, ventralis, rissoi) exhibited a decrease in growth rate only after reaching the Antarctic waters.

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19 METHODS The otoliths analyzed for this study were collected during November-December 1983 and March 1986 as part of the Antarctic Marine Ecosystem Research at the Ice Edge Zone (AMERIEZ) project. Sampling was conducted in the Southern Scotia Sea (60 40W) in the austral spring (1983) and in the northwest Weddell Sea (65S 46W) during the austral fall {1986). These sampling sites are outlined in Figure 4. Pelagic fishes were sampled with both an opening-closing Tucker trawl (9 m2 mouth opening) and a vertically towed, opening-closing Plummet net (1 m2 mouth opening). All fishes were collected between o and 1000m in the open water near the marginal ice zone. Details of the trawling procedures are described in Lancraft et al. {1989). Aboard ship, fishes were identified to species and m easured to the nearest millimeter standard length {SL). The gender of individuals was recorded when possible. The sagittal otoliths of fishes were removed with forceps and stored dry on micropaleontological slides. Sagittal otoliths were prepared for analysis using three sequential microstructural techniques: thin-section grinding and polishing, acetate replication, and scanning electron microscopy {SEM). These techniques have proven useful in

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Figure 4. Sampling areas in the Weddell Sea (A) and the Scotia Sea (B) Numbers are those corresponding to those in T able 1 (adapted from Lancraft et al., 1989). Dashed lines correspond to the ice edge.

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A ' 48W B SCOTIA SEA 0 72. 54 95 10 __ .. _ --5-7-.121 .122 51. 21 ,..... .,20 19 49. 2 7 22 o 30 e17 o 441 114 23 16e 46 WEOOELL SEA 39 37 35 10 ________ ,: 14115 (/) 0 CD 50. ORKNEY IS. 5e 10 s al9 7 goE 35 43 41 I 34 0 46 45 3a 36 U) 42 CD ' 2,4,7 31 ',, /1012 .......-g -----14 0 CD Q) 21

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22 ?iscerning microstructural growth increments in the sagittal otoliths of several species (see review Campana and Neilson, 1985). While these techniques require varying degrees of preparation and time, each can be applied consecutively to the same otolith thin-section from the grind and polish technique to the more elaborate SEM application. Embedding Due to their small size (<2mm) and logistical protocol (e.g. thin-sectioning) sagittae were embedded in a lowviscosity epoxy resin following the hard formula recipe of Spurr (1969). Sagittae were first placed in flat embedding molds with the proximal (sulcus acousticus) side down. The mold cavities were then filled with liquid Spurr while simultaneously holding the sagitta flat with a dissecting needle. After filling, the molds were allowed to polymerize for 24 hours at 65C. This resulted in the sagittae being oriented towards the bottom of the mold cavity. The hardened blocks containing the sagittae were trimmed with an Isomet saw to remove excess Spurr. The cuts were made parallel to the long axis of the sagittae. The flat sections containing whole sagittae were then mounted on modified microscope slides with heated Crystalbond. Slides were modified by mounting an accessory piece of glass to each which further elevated the flat sections. This modification improved the grinding and

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23 polishing process by allowing more flexibility in the degree of pressure applied during grinding. Grinding and Polishing After embedding, the whole sagittae were ground by hand to the mid-sagittal plane with water and wet/dry sandpaper (400, 600, 1500, and 2000 grit) then polished using a polishing cloth and a 0.05 micron gamma alumina slurry. All samples were sonicated between successive grinding and polishing to avoid cross contamination of grits and reduce surface scratches. Sagittae were abraded until a plane through the core was visible and the increments within the larval zone could be enumerated. During abrading three distinct regions became visible. Using the terminology and definitions of Gartner (1991) these were the premetamorphic or larval zone (LZ) postlarval zone (PZ) and postmetamorphic zone (PMZ) These regions are described in the results. At this stage of processing two radius measurements were recorded using an ocular micrometer (Figure 5). The radius of the entire otolith (total radius; TR) was measured from the central core (nucleus; NU) to the outer edge along the posterior axis (40x magnification). The larval zone radius (LZR) was measured from the central core (NU) to the last clear continuous incremen t (metamorphic check) in this region (250x magnification). All micrometer

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Figure 5. Lateral view of Electrona antarctica sagittal otolith, showing the radial axes measured for morphometric analyses and directional axes. TR: total radius, LZR: larval zone radius. Sagitta extracted from a 90mm SL, female (ca. 40x).

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25

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26 measurements were converted to millimeter units. The radii of the sagittae were regressed against standard length. The larval zone (LZ; or premetamorphic zone) was first enumerated using light microscopy. Increment counts were made under oil with a compound microscope coupled to a high resolution video camera and monitor system. All counts were initiated from a well-defined medial increment (i.e. hatch check; 63 Ox magnification) grinding and polishing was within the larval zone. continued to further Then, resolve increments within the accessory primordia (AP) positioned adjacent to the metamorphic check. Accessory primordia, when present, were next enumerated with light microscopy. Each region (LZ and AP) was counted at least three times by 2-3 independent readers. Counts were accepted if readers agreed within a three percent range of error. If counts between readers differed more than 3%, two readers repeated the count together. If repeated counts did not agree, then the sample was not included in the analysis. Light photomicrographs were made of several LZ regions as permanent records of counts and for comparison with other techniques applied to the same sagitta. A subsample of sagittae in which the premetamorphic zone had been successfully enumerated was selected for further processing.

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27 Etching After initial viewing and enumeration of the larval zone and accessory primordia with light microscopy, whole sagittae were further processed for acetate replication and SEM. Whole sagittae were sliced along a frontal plane with an Isomet saw to obtain a flat section containing the entire core region; section thickness varied according to larval zone diameter. The frontal section was selected because of the anteroposterior growth of the sagitta. This growth axis incorporated the entire growth history of the individual and contained the least compressed increments. Sections were rinsed with alcohol, remounted on modified slides, and abraded (grinding and polishing technique) until a plane through the core and peripheral regions was visible and the increments within the postmetamorphic zone could be discerned using light microscopy. Sections were polis hed smooth and then etched using solutions of 1 % HCl (hydrochlori c acid; .12N and ca. pH 2.0-3.0) or 5% EDTA (ethylene diamine tetraacetate; with pH adjusted to ca. 8 0 with NaOH) Etching with weak acid solutions tends to preferentially remove material from the proteinaceous discontinuous zones while EDTA acts as a calcium chelator in the incremental regions. This produces a threedimensional relief that provides consistent patterns throughout the sections. Additionally, etching minimizes microstructural artifacts introduced through preparation

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28 techniques. An array of exposure times were used to determine protocol (30 300 seconds). To discontinue etching, sections were gently rinsed with deionized water, air dried and stored in a desiccator. Macroincrements were enumerated using light microscopy for comparison of etched and non-etched readings of the sections. Photographs of etched sections were made for comparisons and as permanent recordings. A second etching technique, reverse polarization or sputter etching was also explored to determine its usefulness in resolving microincrements in sagittae. This technique had not been previously applied to otoliths, and was attempted as an experimental application from a reasonable theoretical basis. This methodology is a physical (sputtering of single atoms) and a chemical (breaking of chemical bonds) technique that reveals the subsurface cell structure in the SEM. At present, this technique is in its early developmental stages and its application and limitations remain to be investigated. The process is accomplished by ion bombardment using a direct current and an operating gas (Argon) during which the polarity of the anode (stage) and the cathode (aluminum target) are reversed. The ions are accelerated toward the anode with an energy proportional to the voltage applied. The plasma formed is sufficiently energetic to physically etch the sample surface dependent upon the elemental concentration and the strength of its chemical bonding to other elements in the sample. In other words, the most conservative element with

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29 the weakest bonds will be knocked off first by the impinging ions. Frontal sections were polished, sonicated and dried as previously described. Then placed in the chamber of a Hummer Jr. {Technics) sputter coater equipped with an etch/clean function. Exposure (etch) times ranged from 5-15 minutes under a pressure of 50-80 millitorrs (Argon) with 10 milliamperes of current. The resultant surface of the sample rendered a three dimensional relief pattern facilitating viewing with SEM. Acetate Replication and Scanning Electron Microscopy Following etching sections were further processed for acetate replication and viewing with SEM. The acetate peel replicas were made by placing a drop of acetone on a sheet of thin cellulose acetate. The acetone was allowed to evaporate for about 10 seconds. The etched surface of the section was then pressed firmly onto the softened acetate sheet. After drying for about 30 minutes the acetate sheet was peeled off the sections. The replicas were separated and secured to microscope slides using cover slips. The replicas were then enumerated using transmitted light. After acetate replicas were made, sections were secured to metal stubs with double stick tape, coated with goldpalladium alloy in a sputter coater, and examined with the

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30 scanning electron microscope (SEM; 15-20kV). A series of photomicrographs was taken of sections to record the PMZ increments comprising the posterior axis. Montages of SEM photomicrographs were constructed to reproduce the entire growth history of the individual (posterior growth axis) The PMZ microincrements were enumerated from each photomontage. All counts were made along the longest axis (posterior) between the last larval growth increment and the otolith periphery, exclusive of accessory primordia. The posterior axis was chosen for PMZ counts because microincrement clarity and width along this growth axis allowed the most consistent and repeatable counts. Within the PMZ randomly dispersed patches occurred where increments were obscured, although adjacent to these patches increments were distinguishable. To enumerate these unclear areas reproductions of adjacent increments bordering these patches were made on clear overlays. An overlay was then placed over a patch and counts were made from the overlay equivalent to the width of the patchy area. This application allowed for accurate and repeatable estimates of the devoid areas, as increment widths varied < 1 micron in the PMZ. Counts were continued along the posterior axis until the outer edge was encountered. Each pre-and postrnetamorphic zone was counted in triplicate from the nucleus to the posterior edge. Increments were generally enumerated along the same radius, when this was not possible counts were made along adjacent radii by

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31 following clear increments from the main primary radius to a alternate radii. Counts and techniques applied to the same section were compared. Of the three techniques, the one rendering the most reliable and repeatable microincrement counts was selected as standard protocol for the remainder of sagittae analyzed. Data Analysis Life spans were determine d for each sex by addition of the pre-and postmetamorphic zone increment counts. Growth rates were determined by regressing standard length on age in days. All analyses and comparisons were considered statistically significant at the p<. 05 level. First order decisions for determining the best fitting regressions were made using the coefficients of determination (r2 ) The models mos t closely fitting the data were further compared using the mean square e rrors of the residual sums of squares. Growth curves were then tested for significant differences in growth rate between sexes for linear regressions using analysis of covariance (ANCOVA; SAS Institute, 1986). The mean square errors of the residual sum of squares of the growth curves were compared using F values to determine the significance of regressions. If the F values were not significantly different, the intercept (a) and slope (b) for eac h growth curve were compared using Chi-square or a Student's t-test.

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32 Tests for significant departure from normality (KolmogorovSmirnov one sample test for goodness of fit; p<. 05) and homoscedasticity (Bartlett's test for homogeneity of variance; p<.OOOl) were performed (Statgraphics version 6; STSC, Inc., 1992)

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33 RESULTS General A total of 117 (1983, n=98; 1986, n=19) sagittae of Electrona antarctica were examined for this study. All were not readable at every level of examination. The clarity of microincrements within sagittae was inconsistent, consequently, only those yielding concordant and repeatable counts were used for age and growth estimates. A subsample of 60 sagittae were processed for microincrement counts. The total number of individuals examined by tow number, collection date, diel period, depth range, station coordinates, and size are presented in Table 1 The size distribution of postmetamorphic Electrona antarctica from which sagittae were extracted ranged from 23-103mm SL (Figure 6). Several immature individuals were included in this study, as sexual dimorphism is not evident until ca. 4 Omm SL. At this size females have developed infracaudal luminous scales, and males have produced supracaudal luminous scale, a dimorphic characteristic typical of several myctophid species. Length frequency analysis of individuals revealed a distinct change in size between males and females.

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34 Table 1. Spring (1983) and Fall (1986) traw l d ata from AMERIEZ cruises during which otoliths were collected. Tow no. refers to those outlined in Figure 3, N: dusk/night, D: dawnjday, SL: standard length. Tow Date no. 3 14NOV83 5 1 6NOV83 7 17NOV83 8 17NOV83 9 1 8NOV83 10 19NOV83 11 19NOV83 12 20NOV83 14 22NOV83 19 25NOV83 21 25NOV83 22 26NOV83 24 27NOV83 25 27NOV83 28 30NOV83 3 09MAR86 5 09MAR86 7 10MAR86 12 11MAR86 13 12MAR86 1 6 12MAR86 Diel period N D N D D N D N D N D D D N D N N N N N N Depth (m) 0 -760 200-430 100-260 0-1000 300-400 270-400 430-520 0-90 400-900 100-200 310-410 390-530 190-280 350-550 500-980 0-120 0 -150 200-300 40-90 170-260 0 -400 Latitude (S) 6028.4 5929. 8 5836.2 58.1 5826.9 5852. 4 5921.6 5956. 8 5950. 9 6040.9 5939.7 5956.1 6028.2 6028.1 5930. 7 6533.3 6533.5 6533.0 6543.3 6549. 5 6529. 4 Longitude (W) 40 3 3. 9 39.0 3 8. 6 3818.3 37.7 37.6 3720.4 Jr24. 8 3r59.5 38.7 39.3 3918.5 40 2. 9 3948.5 39.7 4 7 28. 6 4 7 3 5 1 4 7 2 5 0 4 ro3. o 4652.2 4631. 2 No. fish SL (mm) 19 37 -97 7 53 -93 4 45 -92 6 27 -91 4 39 80 6 23 -98 7 44 -82 4 72 -99 1 37 3 75 90 13 34 -94 10 34 -99 6 36 103 4 41 101 4 45 -71 3 49 -75 10 51 -92 1 99 2 48 -62 2 49 -99 1 25

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Figure 6. Pooled (1983 and 1986) length distribution of individuals from which sagittae were extracted. Hatched: immatures (n=17), Filled: females (n=34) Open: males (n=66)

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>(.) c: Q) ::::J tT Q) ... u. unsexed (n = 17) D male (n = 34) 15 1 female (n = 66) 10 5 0 ID >I ">I I I D > I I 20-29 30-39 40-49 50 59 60-69 70-79 80-99 90-99 1 00-1 09 Standard length (mm) w 0\

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37 The maximum size of females (43-lOJmm SL) exceeded males (37-83mm SL) by 20 rom. These gender related size differences were reflected in both spring and fall collections. Results from this study corroborate the size related sexual dimorphism previously reported for Electrona antarctica. Size related sexual dimorphism is common to several myctophid species. Methodological Results Due to the intrinsic difficulties (e.g. small size, thickness, and increment width) in observing the microincrements within the sagitta of Electrona antarctica, several experimental methodologies were pursued during this study, which rendered various degrees of success. No single technique was sufficient in resolving microincrements along the entire growth axis. Therefore, a combination of techniques in concert were necessary for consistent and repeatable resolution. A detailed account of the results from these experimental applications is outlined in Appendix 1. At each sequential stage of processing, the clarity of growth increments was enhanced, while macrofeatures became less pronounced. To summarize, the larval zone (LZ) and accessory primordia (AP) were enumerated using transmitted light under oil, and counts of the perinuclear (PZ) and postmetamorphic (PMZ) zones were made from SEM photomontages.

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38 Otolith Morphology: External The overall shape of the sagittae of Electrona antarctica is a domed ovoid. They are anteroposteriorly elongated with a rounded slightly bulbous posterior end and a narrower rounded anterior end. The entire perimeter is smooth with the rostrum, antirostrum and excisura ostia being rounded. The central part of the sagitta overlying the core region is thickest having convex distal and concave proximal surfaces (i.e. medially elevated, laterally convex). Sagittae become thinner and more translucent along the anteroposterior periphery. The smallest sagittae from fish < 40 mm SL were thinner, flattened and displayed an elliptical symmetry. As sagittal size increased (fish > 40 mm SL) they grew successively thicker centrally and more elongate anteroposteriorly. Sagittae of male and female individuals of small, intermediate, and large size ranges are represented in Figure 7a, b and c. No conspicuous external differences were observed between males and females, except for overall size in the largest sagittae belonging to females. A total of 117 sagittae were e xamined morphometrically. Two radii measurements were made from at least one sagitta of each pair; the total radius (TR) and larval zone radius (LZR). The pooled radii measurements taken along the anteroposterior axis ranged from 0.425-1.9 mm for the TR and from 0.114-0.165 mm for the LZR. Radii measurements recorded for individuals

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Figure 7. External morphology of sagittae of immature, male, and female Electrona antarctica fro m s mall, intermediate and large size ranges. F: female, and M: male. All lengths are reported as standard length in mm. All photographs taken at ca. 1.5x. A) 48mm (F), 45mm (M), 53mm {F), 56mm {M) B) 75mm (F), 75mm (M) C) 90mm (F), 98mm (F) lOlmm ( F)

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4 0

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41 by size and sex are s u mmarized in Table 2. Morphometric measurements varied between males and females. Application of Tukey's Multiple Range Analysis (Statgraphics1 Corp.) of means revealed significant differences in pairwise tests between mean standard length and total radius 1 while larval zone radius was not significantly different between sexes (Table 3) Regression analysis of standard length on otolith radius showed a strong linear pattern for pooled and unpooled data. The linear relationships for pooled and unpooled data are illustrated in Figures 8 and 9. The regression equations for the pooled and unpooled data are summarized in Table 4. Results of significance testing (ANCOVA's) of all regressions revealed a significant difference in slope (p<.04) and intercept (p<.0 2 ) between males and females for standard length on total radius (SL /TR). Testing for standard length on larval zone radius ( S L / LZR) showed no significant differenc e in slopes (p<.56) although the difference in intercepts (p<.0005) was significant between sexes. Otolith Morphology: Internal For the following descriptions of internal morphology, the terminologies for otolith microstructure defined by Campana and Neilson (1985) and Gartner ( 1991) are used as consistently as possible. For clarification 1 the term "growth

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42 Table 2. Morphometric data recorded from sagittae of Electrona antarctica. N : number of individuals, U: unsexed, M: males, F: females. All measurements are in millimeter (mm) units. STANDARD RANGE RANGE LENGTH N SEX TOTAL RADIUS LARVAL ZONE 21 30 3 u 0.425 0.550 .126-.135 31 40 8 u 0.675 0.750 .120-.144 1 M 0.725 .135 5 u 0.800 -1. 200 .129 -.156 41 50 6 M 0.775 1.000 .135-.156 7 F 0.850 -1.125 .120 -.150 51 60 5 M 1. 025 -1. 200 .120 .144 13 F 0.975 -1.175 .120 -.165 1 u 1. 375 .126 61 70 10 M 1.125 -1. 375 .120-.150 4 F 1. 200 -1. 325 .132 -.14 7 71 80 6 M 1. 462 1.600 .124 -.135 11 F 1. 300 -1. 500 .120 -.150 81 90 6 M 1. 500 -1.650 .120 -.144 10 F 1. 375 -1. 700 .120 -.156 91 100 19 F 1. 475 -1. 900 .120 -. 162 101 -110 2 F 1. 787 -1. 725 .126 -.135

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43 Table 3. Results of Tukey's multiple range test of means for standard length, total radius and larval zone radius by sex for Electrona antarctica. Means with *'s under the letters A, B and C were not significantly different in pairwise tests. Data are pooled for all fish collected in 1983 and 1986 (n=117). N:total number of individuals, SL: standard length (mm), TR: total radius (mm), LZ: larval zone radius. Sex N SL Mean A B C TR Mean A B C LZ Mean A B C unsexed 17 38.118 0.776 0.131 males 34 63.441 1. 258 0.133 females 66 74.924 1. 405 0.136 Table 4. Linear regression equations of standard length (Length in mm) on sagitta total radius (Radius in mm) for Electrona antarctica. Sex pooled unsexed females males Regression equation Length = -5.65 + 56.56 (Radius) Length= 4.40 + 43.46 (Radius) Length = -9.35 + 60.00 (Radius) Length = 1.80 + 49.01 (Radius) n 117 17 66 34 0.938 0.929 0.911 O.g!j)

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Figure 8. Linear regression of standard length on sagitta total radius for pooled data of all individuals, n=ll7.

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4 5 --E E Cl) u ..... "ffi 0 '0 Q) 0 0 V') a. 0 0 ....... (ww) tnBual

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Figure 9. Linear regression of standard length on sagitta total radius for unpooled data arrayed by gender. unsexed: diamonds, n=17 females: circles, n=66 males: triangles, n=34

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0 N "'0 (].) X (].) en c: :::J --en Q) (\S E Q) -47 -E E en :::J -:0 (\S -0 -81 16 E V') 0 0

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48 increment11 termed 11ring11 by Gartner (1991) refers to a bipartite structure, composed of a calcified incremental zone and a proteinaceous discontinuous zone, usually formed over 24hr. (Mugiya et al, 1981). The macrofeatures and internal morphology of the sagittae of Electrona antarctica are shown in Figure 10. Three distinct regions were apparent within each sagitta; the larval zone (LZ) and accessory primordia (AP), postlarval zone (PZ), and postmetamorphic zone (PMZ). Assumptions A priori, two assumptions regarding the present research were made as follows. The first assumption was that one growth increment is deposited per day. This has not been validated, but the increments appeared analogous to those in other species for which primary growth increments have been validated as daily. The inter-increment distances further supported this assumption. A second assumption was that sagitta increment size was a function of growth rate. The correlation between otolith size and fish size is a foundational assumption for most age and growth studies, and the findings for antarctica have been shown for other myctophids as well.

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Figure 10. Overall view of internal morphology of sagitta from Electrona antarctica (82mm, female) . Transmitted light lOOx. LZ: larval zone, : accessory primordia, PZ: postlarval zone, PMZ: postmetamorphic zone.

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;11,' ... .. . r-tJ ... ,, '( . c .. \ .J' ... < 5 0 "\. .,

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51 Larval Zone i1Zl The internal morphology of the larval zone is represented in Figure 11. The LZ or "nucleus" was off-center anteriorly within the sagitta and encompassed a single primordium. The larval zone included the core out to the metamorphic check. The metamorphic check (MC) usually defined the last continuous circular increment within the larval zone. LZ counts were initiated from a well-defined medial increment or "hatch check" (HC). Frequently, increments could be seen within the HC, but increments were not consistently present. Therefore, the HC increment was determined to be the most consistent temporal structure present for initiating counts. LZ increments were usually quite clear ranging in width from 2 microns towards the center to 6 microns near the periphery at the MC. Daily increments within the LZ were generally clear, resulting in an 80% success rate in repeatable counts. Subdaily or nondaily (Geffen, 1982) increments were also visible throughout the larval zone and generally ranged from 1-2 increments juxtaposed to daily increments. Subdaily increments were not included in estimates of lifespans. Both I daily and subdaily increments are shown in Figure 12. Distinction between daily and subdaily increments, although not totally objective, was made based on relative optical density and continuity after shifting of the focal length.

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Figure 11. Entire larval zone with metamorphic accessory primordia visible. NU: accessory primordia. Transmitted light 40x.

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Figure 12. 11Daily11 and subdaily increments within the larval zone of Electrona antarctica. NU: nucleus. Transmitted light 630x.

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55

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56 Another characteristic feature of the larval zone which occurred frequently but not consistently was the accessory primordia (AP). These structures projected radially from the metamorphic check as outcroppings or extensions of the LZ clearly demarcated from the PZ (postlarval zone) Increments radiated from the AP foci towards the distal edge of the structure and ranged from 3-10 in number. Although the presence and formation of AP and their significance have been previously described for several myctophid species (Linkowski,1991; Gartner, 1991), the significance of these structures in Electrona antarctica is unclear and was not further considered during this study. When present, counts of the AP were included with estimates of the total lifespan. A total of 60 otoliths were successfully enumerated for estimates of larval stage duration. Mean increment number varied with counts ranging from 27-48 increments. The mean counts of the LZ made independently by three readers are summarized in Appendix 1 a, b and c. Counts did not vary significantly by sex (Tukey's test; p < .05). No correlation was apparent between the number of increments in the LZ and standard length of individuals (Figure 13). Postlarval zone lEZl The PZ is outlined in Figure 11. This portion of the sagitta was extremely dark suggesting a high concentration of

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Figure 13. All increment counts in LZ (n=60) regressed against standard length of individuals. 1983=triangles 1986=circles.

PAGE 71

59 protein matrix and further suggesting a slower growth phase. The PZ was nearly impermeable to transmitted light, only broad bands ranging from 5-12 were visible. These broad bands may be analogous to the PZB's described by Gartner {1991). The width of the PZ ranged from 1.38-1.55 mm, nearly the same as the LZ radius. The increments within this region were elucidated using SEM, however, the broad bands were no longer visible at high magnifications. Within this region increments were often indistinct or incongruous. When the increments could not be successfully (5% repeatability) enumerated, an average increment count of 50 (range 38-60) calculated from readable sagittae was added to the total. The effects of estimating this portion of the growth axis accounted for <5% of the total lifespan. Postmetamorphic zone CPMZ) The postmetamorphic zone comprised the region from the terminal increment of the PZ to the sagitta periphery or outer edge. The PMZ was abruptly demarcated by an apparent transition in elemental composition. Under transmitted light the dark, nearly opaque, proteinaceous PZ sharply contrasted the contiguous, more translucent, predominantly aragonitic matrix of the PMZ. Within the PMZ a regular pattern of alternating continuous and discontinuous zones defined the remainder of the growth axis. When observed with SEM the

PAGE 72

60 incremental (accretion) zones are broader and only lightly etched, while the discontinuous areas are narrow and deeplyetched (Figure 14). PMZ increments were strikingly consistent with widths ranging from 0.8-1.4 microns (Figure 15). Increments observed in frontal sections began as narrowly ushaped structures then became elongated and broadly U-shaped posteriorly toward the outer growth edge. A total of 33 sagittae comprising 20 females, 11 males and 2 unsexed individuals, were enumerated for estimates of postmetamorphic age and growth. All PMZ increment counts are summarized in Appendix 3. As in the LZ, finer scale nondaily increments were observed throughout the PMZ. These structures were not as consistent as in the LZ and usually comprised only one increment positioned between "daily" growth increments. These substructures were not further considered for this study. Other PMZ Features The continuity of structures other than "daily" growth increments was quite pronounced in the sagittae of Electrona antarctica. One such feature was a striking pattern of growth discontinuities possibly of a "lunar" or "monthly" temporal nature (Figure 16). These structures were observed throughout the PMZ in frontal sections and whole sagitta. High magnification observations with SEM revealed that these

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Figure 14. Low and high magnification SEM views of an etched frontal section showing the three-dimensional relief pattern characterizing samples. Arrows mark the narrow and deeply etched discontinuous regions. Magnification 810x (left side) and ca. llOOx (right side)

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62

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Figure 15. SEM photomontage of the PMZ illustrating the daily increments within this zone. 68mm SL, female. Magnification 1005x.

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64

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Figure 16. Low magnification SEM view of an etched frontal section showing the internal structures medially towards the periphery. The structures marked with arrows are possible "monthly" or "lunar" temporal signatures. Magnification 200x.

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66

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67 increments etched very deeply and appeared as dark, broad discontinuous zones, suggesting a proteinaceous composition. No growth increments (e.g. daily) were observed within these checks. The lunar or monthly timing is suggested based on enumeration of the growth increments lying between these marks (Figure 17) A range of 28-34 increments were counted contiguous to these checks, and the structural continuity of adjacent increments was maintained while increment widths did not appear to change. A second growth pattern was observed under a dissecting microscope with reflective light. Alternating opaque and hyaline bands were visible within the PMZ (Figures 5 and 8). These bands were analogous to those described by Linkowski (1987) which he concluded were "annual" temporal marks. For this study these structures are referred to as "false" annual growth bands. Following the first stages of sagittal processing (grinding and polishing), these features disappear and are not observed within the sagitta at any subsequent level of examination. Age estimates calculated using these growth marks were similar to Linkowski's published estimates for Electrona antarctica (8 years). Growth Rates and Life spans An initial growth curve for Electrona antarctica was estimated from pooled data (Figure 18). Linear growth models

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Figure 17. High magnification SEM view showing the growth increments juxtaposed to two "monthly" time marks. Magnification 2,200x.

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69

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Figure 18. Linear model of growth from pooled data for all Electrona antarctica.

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71 0 0 II") 0 Q) 0 II") N -(/) >.. (\j "0 0 C/) II") -I" c:: (]) E (]) ..._ 0 1:J c: (]) 8 0 II") 0 a. 0

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72 described trends in the data well, and reflected the individual growth trajectories best. It is relevant at this point to emphasize that the growth rates estimated for this study are for sexually dimorphic individuals >40mm SL. The entire lifespan of E. antarctica is not reflected in these estimates. Model selection was determined as the one accounting for the greatest variability and having realistic growth maxima parameters. The growth model selected for this study is represented by the linear equation: Length = a + b (Age) where length is the standard length in mm; a is the estimated intercept; b is the estimated slope, and; age (in days) is estimated from growth increments. Separate growth curves were generated for each gender (Figure 19). The best fit for individual growth rates during this size-specific (40-lOJmm SL) growth stanza was a linear model. Significance testing using ANCOVA showed a significant difference in growth rates for females and males (slopes p <.007 and intercepts p <.Ol). Females continued along the established trajectory, while males diverged slightly from this pattern. Female growth rates appeared to be relatively constant at .07mmjday, while males showed slower growth rates averaging .05mmjday. The growth equations for pooled and unpooled data are presented in Table 5 Lifespans were determined for each sex by addition of the LZ, PZ and

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Figure 19. Growth rates estimated for male and female Electrona antarctica.

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Table s. Sex pooled females males Equations for the overall growth individual growth curves for male Electrona antarctica. Growth Equation 8.869 + 0.063 (Age) 2.225 + 0.072 (Age) 16.618 + 0.051 (Age) n 31 20 11 75 curve and and female 0.946 0.985 0.987 PMZ regions. The overall ages ranged from 403 to 1355 days for Electrona antarctica over the size range of 40mm to 103mm SL. Males exhibited a slightly shorter lifespan than females. The range and maximum for males were from 403 to 1254 days, while females showed a longevity maximum at 103mm SL of 1355 days.

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76 DISCUSSION In the present study microincrements (primary growth increments) within sagittae are assumed to represent the day by day developmental history of Electrona antarctica. The depositional patterns of the otoliths are indicative of an individual's growth and its environmental conditions. Major and minor events are reflected in the characteristic "banding" of the sagittae, including monthly signatures that reflect disruptions cued by lunar cycles, and transition zones representing hatching and metamorphosis. Annual deposition patterns have not been detected in the otoliths of Antarctic fishes (Radtke and Targett, 1984; Radtke and Hourigan 1990; Radtke et al., 1989}, perhaps due to the lack of distinct annual periodicity in hydrographic conditions (Radtke, 1984). There is a striking regularity of microincrements within the sagitta of antarctica; the lack of distinct annual rings leaves the investigator with daily rings as the best (and only) means of estimating individual age. This trend may apply for other mesopelagic fishes inhabiting the Southern Ocean (Greely, personal observation; Linkowski, 1987). There is general agreement that deposition of daily increments is governed by an endogenous rhythm, but that ring structure can be influenced by exogenous factors e.g.

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77 temperature, light, and pH (cf. Campana and Neilson, 1985). Gartner ( 1991) proposed that for Gulf of Mexico myctophid species, ring (increment) formation may be linked to activity levels and that light might be an indirect zeitgeber. Radtke et al. ( 1989) used the Antarctic nototheniid, Trematomus newnesi, to show that daily increments are deposited even during the winter months, despite considerable variation in photoperiod during this season. The solar dark-light cycles at high latitudes (60-70 ) are characterized by long periods of daylight ( 18-2 4 hours) during summer followed by long periods of low light intensity (0-6 hours) during winter. In an elegant study, Townsend and Shaw (1982) tested the importance of a diel light-dark cycle in the deposition of daily increments by examining blue whiting collected in summer above and below the Arctic circle. Results showed no modification of daily increment formation in fishes exposed to 24 h of continuous daylight above the Arctic circle. Thus, all high latitude species that have been examined possess an endogenous rhythm of ring deposition independent of an environmental light cue. This almost certainly explains the daily increment formation observed in Electrona antarctica. Previous studies Although the cosmopolitan importance of myctophids has been well documented, age and growth studies based on

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78 microincrements in otoliths have primarily focused on tropical and subtropical species {Gj0saeter 1987 ; Young et al. 1988; Gartner 1991; Giragosov and ovcharov, 1992). Whereas higher latitude species, because of the presumed seasonal effects on growth have been aged using annual growth increments. Linkowski {1987) observed primary growth increments in the otoliths of two subantarctic species, E. rissoi and E. (Metelectrona) ventralis. Maximum ages in tropical-subtropical species ranged from 1. 0 to 2 0 years and in subantarctic species from 1.5 to 2 0 years. The first published age data for E antarctica was contributed by Rowedder (1979a), who found age determinations impossible using otoliths or opercula. Consequently, based on the trimodality of the length frequency distribution {n=1373; 23-104mmSL) he concluded that E antarctica reached a maximum age of 3 years, assuming that each mode represented a single year class. Miya et al. {1986) found similar modality in size-distribution data (n=82) for E antarctica collected in the Southern Ocean south of Australia. Miya et al. (1986) proposed that E antarctica matured in 2 years at 70-85mm SL and reached a maximum age of 3 years. The estimates of age presented by Rowedder and Miya agree nicely with the results of the present study, though they are subject t o the problems associated with length frequency analysis for age and growth determinations. Namely, as older fishes asymptotically approach a maximum size, an all-inclusive mode at the largest

PAGE 91

79 size class can mask the presence of several year classes. Before the present work, no study had been successful in using otolith microstructural techniques for age determinations of Electrona antarctica. Linkowski (1987) was unsuccessful in finding primary growth increments in the sagittae antarctica. Therefore, he used reflected light to make direct counts of zonal macrostructures, alternating hyaline and opaque zones, assuming that one opaque and a consecutive hyaline ring represented an annual time mark. Subsequent results using external features suggested males obtained 8 and females 11 years of age. This resulted in maximum ages nearly three-fold greater than estimated in the present study. The findings of the present study suggest that the large deviation reflected in Linkowski' s estimates for Electrona antarctica are the consequence of enumerating false annual time marks resulting in an overestimation of age. Use of presumed annual time marks for age estimations has resulted in an overestimation of age for other mesopelagic species (Methot, 1981; Lancraft et al. 1988; Young et al. 1988; Gartner, 1991). Examination of the internal morphology of the sagittae revealed that no annual deposition patterns were visible, suggesting that these externally visible features were the result of differences in compositional densities within the three-dimensional structure of the whole sagitta. Although numerous rhythmic discontinuities (possible monthly

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80 time marks) were observed internally within sections they could not be correlated to external features. This hypothesis was further tested by measuring the distance between false annual marks using reflective light microscopy, and comparing the s ame regions (equi-distant) internally. Several monthly checks (3-6) comprised the test regions but never did the values approach 12 monthly checks, which would support the interpretation of those external features as representing annual time marks. Nor did the number of primary growth increments within those regions approach 365 in number. I therefore suggest that the use of microincre m ents to estimate the age and growth rates of E antarctica is more reliabl e than presumed annual time marks. Validation Daily growth studies for Antarctic fishes have focused almost exclusively on the dominant, demersal, coastal members of the suborder Notothenioidei (North et al. 1980; Townsend 1980; Radtke 1984; Radtke and Targett 1984; Radtke et al. 1989; Radtke and Hourigan 1990). Two studies confirmed that the microincrements found within the otoliths of Antarctic fishes were deposited daily (Radtke et al. 1989; Radtke and Hourigan, 1990). Growth increments were validated by use of t etracycline incorporation into sagittae. The first validation of daily increment formation in a

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mesopelagic fish was contributed by Gartner (1991). 81 Using marginal increment analysis, he verified daily increment formation for three myctophid species inhabiting the Gulf of Mexico. This work stands alone as the only study to validate daily growth increments in a midwater fish. Validation of daily increment deposition in the sagitta of Electrona antarctica may also be obtained by marginal increment analysis, as in Gartner (1991). Marginal increment analysis was not attempted in the present study, due to limitations of the sampling regime during previous cruises to the WeddellScotia sea region. However, an attempt to rectify this shortfall is underway as the result of a subsequent cruise during the 1993 austral summer. Sagitta Morphology The PZ structure described for Electrona antarctica is analogous to reg ions described for other myctophids (Gj0saeter, 1987; Ozawa and Pefiaflor, 1990; Gartner, 1991; Linkowski, 1991). Two interpretations of this zone have been proposed. Gj0saeter described this entire region as a metamorphic check, alternatively, Gartner suggested this zone reflected metamorphosis and a subsequent habitat shift. Certain myctophid species as premetamorphic larvae begin a downward migration from warm, well-lit, surface waters towards colder, darker, mesopelagic depths. The coupling of

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82 metamorphic transformation and extreme change in habitat induce a metabolic response recorded in the sagitta. Gartner's theory of habitat shifting seems plausible for antarctica, based on increment counts and larval distribution patterns. In contrast to Gulf of Mexico species the direction and timing of the habitat shift for Electrona antarctica differs, in that eggs and l arvae remain at depth and migrations to shallower waters begin as late juveniles. Efremenko {1985, 1987) suggested year round spawning of Electrona antarctica in the deep, "warm" water masses from 200-500m and between 500-1000m, where annual hydrological conditions are constant (e.g. temperature, salinity, and oxygen concentrations). In the present study year round spawning of antarctica is evident from backcalculated estimates of birthdates (Figure 2 o) South of the Antarctic convergence peak spawning is probably in summer and autumn when the max imum number of eggs (1.2-1.4mm diameter) and larvae are found (Efremenko, 1985). Pelagic eggs, larvae and postlarvae are abundant in winter below 200m. Post-larval individuals appear to experience a growth hiatus as reflected in the dense protein matrix of the PZ suggesting a much slower growth phase than larvae. This slower phase of growth may correspond to remaining at depths >200m, as has been suggested for the presence of the PZ in several Gulf of Mexico species by Gartner {1991). Juveniles

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Figure 20. Backcalculated birthdates estimated for the 32 individuals used for age and growth analysis.

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SPRING 80 11 I II I Ill I I I I II I I I I I Ill I II II I Ill I I I I I I I I II, WIN1ER801 t I I I I I I I t0e0e0a4s I I r0e0t0e0t I I I I I I I t0t $ t I I :0 : I s0r43 .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. SUMMER 80 1 .......... I I I I I I I I I I I SPRING 81 ______ ............................ .. AUTUMN81;._. .......................... ... SUMMER81,_ ............................ ... SPRING 82 p I I I I I I I I I I I I Z Z I I I I I I I I I I I I I I a WINTER 82 ,, z z z z z z z z a AUTUMN 8 2 I I I I I I I I I 7 I z I I I z I z I I I I z I I z I I I I I SUMMER 82 I I I I I I ? I z I z z I z z z I I I I I I I I I I I I I I I I z I z I I z I z I ::J AUTUMN8 3 SUMME R83 0 I 2 3 4 5 F r eq u ency D fL) 0 1983 1982 1981 1980 00

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85 possibly begin regular diel migrations a couple months after metamorphosis (mean larval duration= 40 days, mean PZ duration= 50 days, total LZ+PZ= 3 months). This change in activity level and habitat (upper 200m) is possibly reflected in the PMZ of the sagitta. growth phase evidenced The PMZ pattern suggests a faster by increased calcium carbonate deposition and increment widths. Egg development and size-dependent vertical distribution data for early life stages of Electrona antarctica are needed to substantiate this thesis. The monthly timemarks described in the present study may be analogous to those described for other species in which distinct annual rings are lacking (Campana and Neilson, 1985; Panella, 1974, 1980; Radtke and Targett, 1984; Radtke et al., 1989). The formation of a regular hyaline structure corresponding to a lunar month was observed in the tropical myctophid, Myctophum nitidulum (Giragosov and Ovcharov, 1992). Physiology/Bioenergetics Myctophids are a dominant component of all mesopelagic faunas and subsequently play a major role in the energetics of oceanic ecosystems. Electrona antarctica is the dominant mesopelagic species in the Southern Ocean in both abundance and biomass (Rowedder, 1979b; Hulley, 1981; McGinnis, 1982; Lancraft et al. 1989, 1991) The rates of physiological processes (respiration and composition) reported for

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86 antarctica are comparable to non-polar myctophids (Torres and Somera, 1988a,b; Donnelly et al., 1990). The characteristics unique antarctica are its higher lipid content, narrower diet, larger size, and longevity. A preliminary energy budget was estimated for Electrona antarctica. A mid-sized (72mm SL), 2. 5-3 year old female weighing 8.59g (wet weight; WW) was selected for a first order approximation. Several feeding studies (Rowedder 1979b; Hopkins and Torres, 1989; Kozlov and Tarverdiyeva, 1989) have described a size-dependent diet shift. As body size increases the contribution of copepods (Calanus prooinguus and Metridia qerlachei) in the diet declines while the number of krill (Euphausia superba) increases. Due to insufficient data for the energy levels of prey items other than krill, a large krill consuming female was used for computations. This restriction should allow a more biologically meaningful budget. Four components comprise the basic model: INGESTION = METABOLISM + GROWTH + EXCRETION Ingestion rates for myctophids are typically size specific and range from 2-5% of the total body wet weight (Clarke, 1973; Hopkins and Baird, 1985). From diet analysis Rowedder ( 1979b) calculated an annual ration of 87gDW (21 times BW) for Electrona antarctica taken during summer. For the present study ingestion was estimated by calculating two

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87 daily rations for 2% body weight (BW) and 5% BW. Using the mean caloric value, 97.1 kcal calculated for krill (30-53mm SL; Torres et al., in press) and the 2% (0.123g WW) and 5% (0.309g WW) body weights annual ingestion rates were estimated as: I (2%) = (0 .123g WW) X (97 .1 kcal 100g-1 WW) x 365 MINIMUM INGESTION = 43.59 kcal y1 and I ( 5% ) = ( 0 3 0 9 g WW) X ( 9 7 1 k cal 1 0 0 g -1 WW) X 3 6 5 MAXIMUM INGESTION = 109. 51 kcal y-1 Metabolism as the rate of energy expenditure was measured using the rate of oxygen consumption determined by Torres and Somero ( 1988a) The average o xygen consumption rate for Electrona antarctica at 0. 5 C was 0. 042 0 0003 (ml 02 g-1 WW h-1 ) Multiplying the weight of a mid-sized individual (72mm = 6.17g WW; range 40-103mm SL) by the oxygen consumption rate, time, and an oxycaloric equivalent of 4. 63 kcal 1-1021 the energy consumption due to respiration was calculated. Two metabolic rates, standard and active, were calculated to account for vertical migrations (8h) and non-migrating (16h) times. The average oxygen consumption rate (0. 042ml 02 mg -1 WW h-1 ) was used for non-migrating hours and the maximum measured rate ( o. 069ml 02 mg-1 WW h-1 ) was selected for migratory hours. Metabolism was estimated as: (0.042ml 02 g-1 WW h -1 ) x (6.17g) x (16hr) X (4.63 kcal L-102 ) X (1000) X (365)

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M (non-migration) = 7. o kcal y 1 (0.069ml 02 g1 WW h"1 ) x (6.17g) x (8hr) X (4.63 kcal X (1000) X (365) M (migration) = 5. 7 6 kcal y1 TOTAL METABOLISM= 12.76 kcal y1 88 Growth was estimated by using the mean caloric value of 161.7 kcals 10og1 WW ( 140-184kcals 10og1 WW) as determined by Donnelly et al. (1990) for Electrona antarctica 29-101mm SL1 over 3 seasons. To estimate growth in % BW d-1 1 Rowedder' s ( 1979a) standard length (mm) jwet weight (g) equation was applied. The annual energy for growth was determined by multiplying the mean caloric value (161.7kcal 100g-1 ) by the annual body weight increase between year 2 and 3. From the growth model estimated in the present study, a 2 year old female is approximately 2.80g ww at 55mm SL and a 3y female is 8.59g at 81mm SL, yielding a weight increase of 5.79g. Annual growth was: ( 5. 79g) x ( 161. 7kcal 10og1ww) GROWTH = 9. 36kcal y1 The excretion value for Electrona antarctica was estimated to be 27% of the ingestion in kcal day' 1 which is considered a reasonable excretion value for zooplanktivorous fishes (Brett and Groves, 1979). The estimated annual excretion rate was:

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E = (.27) X (I(2%) = 43.59kcal) MINIMUM EXCRETION = 11.76kcal and E = (.27) X (I (5%) = 109.51kcal) MAXIMUM EXCRETION = 2 9. 5 7kcal y-1 89 The amount of energy lost due to heat (HI) was estimated as 14% of the gross ingested energy (Brett and Groves, 1979). These values were determined to be: HI= (.14) x [I(2%) = 43.59kcal] MINIMUM HEAT LOSS = 6 .10kcal y-1 and HI= (.14) x [I (5%) = 109.51kcal] MAXIMUM HEAT LOSS = 15. 33kcal y-1 Thus, the total energy lost to excretion and heat from ingested energy was: MINIMUM HI+ E = (6.10 + 11.76) = 17.86kcal y-1 MAXIMUM HI+ E = (15.33 + 29.57) = 44.90kcal y-1 When these estimated values are applied to the model at minimum and maximum ingestion levels the components of the energy budget equate to: MINIMUM VALUES: I ( 4 3 59) [HI ( 6 10) + E ( 11. 7 6 ] = M ( 12 7 6) + G ( 9 3 6) Resulting in a starting ingestion value of 25. 73kcal y-1 and an energetic expenditure in metabolism and growth of 22 .12kcal y-1 accounting for 86% of the hypothetical ingested energy at minumum ingestion rates.

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90 MAXIMUM VALUES: I(109.51) -[HI(15.33) + E(29.57) = M(12.76) + G(9.36) Resulting in a starting ingestion value of 64.60kcal y1 and an energetic expenditure in metabolism and growth of 22 .12kcal y1 accounting for 34% of the ingested energy at maximum ingestion rates. There is a clear surplus of energy available to Electrona antarctica individuals during periods of maximum feeding. Although the above budget does not account for reproduction, it is assumed to constitute a small fraction of the total body calories. It is likely that there is a seasonal oscillation in ingestion rate (e.g. winter months) that results in the yearly ingestion being closer to 2% than 5%. Growth rates are direct (no accounting for reproduction), metabolism is direct, ingestion indirect, excretion indirect. Size-dependent differences in water, protein and lipids have been cited; as size increases a concurrent decrease in water and protein with a substantial increase in lipids occurs. Lipids are mainly wax esters (Reinhardt and VanVleet, 1986). Ecology Electrona antarctica is the only lanternfish endemic to the Southern Ocean, being geographically restricted south of the Antarctic Convergence. Characteristic of many myctophid species, antarctica is a strong diurnal vertical migrator,

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91 with peak abundances occurring between 0-JOOm at night and 650-900m during the day (Lancraft et al., 1989). Seasonal variations in vertical distribution are most pronounced during wintertime. Depths of maximum nighttime (100-400m) and daytime (>1000m) abundances are greater in winter than during the fall and spring (Lancraft et al., 1991). Although shifts in the minimum depth of occurrence (MOO) are evident these population shifts do not appear to impact the growth rates of Electrona antarctica, as evidenced by the continuity of interincrement widths throughout the PMZ (i.e. there is no obvious decrease in calcium carbonate deposition nor a protein-rich overwintering band). A concurrent shift in the MOO (below 200m) of zooplankton biomass species, exclusive of Euphausia superba, occurs in winter, although there is little seasonal difference in prey biomass (Lancraft et al., 1991). It would therefore appear that the coupling of abundant prey and high lipid reserves affords antarctica a continuous growth rate throughout the year. Concurrent with population shifts in prey items there is a decrease in the number of krill consumed in winter (Lancraft et al., 1991), which could easily be supplemented by use of lipid stores. In conjunction with increased lipids in late autumn and early winter, energy levels increase by approximately 15% from spring to fall and from fall to winter (Donnelly et al., 1990). Furthermore, the estimated energy budget predicts a surplus of available energy for growth and metabolism even at minimum ingestion rates,

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92 probably occurring in winter. Linkowski (1987) suggested that the growth parameters estimated for four Antarctic and subantarctic species of Electrona conformed well with their patterns of distribution. He proposed a slower growth rate for species occupying colder Antarctic waters throughout their life cycle (e.g. antarctica) while those which migrated to Antarctic waters as adults carlsbergi, rissoi and ventralis) exhibited a decrease in growth rate only after reaching the Antarctic waters. The present study partially supports this explanation, in that antarctica does have a slower growth rate and greater longevity than migrating species rissoi, 1.5 years ventralis, 2 years), but the magnitude is far less than predicted by Linkowski (8-11 years; Figure 21). An antarctica that lives for 8-11 years would exhibit a growth pattern quite similar to other high latitude myctophid species aged using concentric zones in otoliths assumed to correspond to annual growth. Concurrent with this longevity is an extremely slow growth r ate that is difficult to explain ecologically as an adaptive advantage, given the central role of antarctica trophodynamically In contrast, the growth pattern proposed in this study suggests a 3-4 year longevity. A shorter life-span is physiologically reasonable based on metabolic rates, proximate composition, and seasonal adaptations previou sly reported for Electrona antarctica (Torres and Somero 1988a,b; Reinhardt anc

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Figure 21. Growth rate estimates for antarctic and subantarctic myctophids. Gymnoscopelus nicholsi ( ----) and Electrona antarctica ( ) determine d from concentric zones in otoliths assumed to correspond to 1 years growth. Electrona antarctica (--; present study) and Electrona ventralis (--) determined from daily growth increments. Values taken from Linkowski (1985, 1987).

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160 ,........, E 140 E 120 """'-"" .t: ..., 100 C) r:: Q) 80 -c J.. 60 m -c r:: m ..., en 20 0 G. nicholsi ..p E. ventralis .,.. <" # E. antarctica l''.,' (present study) I ""* .,-E. antarctica ,?; # / Ji # .,. l I' .,. / i * # / I / I / 012 3 4 56 7 8 9 Estimated age (years) \D

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95 VanVleet, 1986; Donnelly et al. 1990). The size-dependent energetic packaging of food resources, including increased lipids, and continuous, although slower, growth rates all contribute to the success of Electrona antarctica as the endemic species within the Southern Ocean. Co mpared to non-polar myctophid species for which daily ages have been determined, antarctica has a slower rate of growth (Table 6) I t is the longest lived and largest of the myctophid species where ages have been determined using microincremental analysis. Without daily growth estimates for other high latitude and temperate species, any comparisons or trends T a b l e 6 Maximum age estimates for 6 tropical-subtropical and 2 subantarctic myctophid species based on primary growth increments. ( Gartner 1991; b Gj0saeter, 1987; c Young etal. 1988; d Linkowski, 1987). Species Benthosema suborbitale" Diaphus dumerilli" Lepidophanes guentheria Benthosema fibulatumb Benthosema pterotumb Lampanyctodes hectorisc Electrona rissod Electrona ventralisd Maximum Standard Length 33mm 63mm 65mm 100mm 50mm 73mm 72mm 110mm Maximum Age 10 11 months 18 -24 months 12 15 months 12 months 9-12 months 36 months 18 months 24 months

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96 characterizing the growth pattern exhibited by antarctica are precluded. Generally, temperate to high latitude species are slower growing and larger than warmer water species. Seasonal variations in the growth rates of cold water species suggest that differences in growth rate may be related to hydrographic factors as well as life-histories (Gj0saeter and Kawaguchi, 1980; Weatherly and Gill, 1987). Electrona antarctica is the end-member species in the continuum of vertically migrating myctophids that extends from the equator to the polar circle. Its growth rate is consonant with all other myctophid species that have been examined using microincrements to determine age (refer to Table 6). In addition, a simple energy budget constructed using the best information available suggests strongly that more than enough prey resources are available to support the growth rate observed. The present data, in conjunction with that of previous studies suggests that growth rates of mesopelagic species are far higher than previously thought (Figure 22). Relative to shallow-dwelling, temperate, coastal pelagics, antarctica exhibits an initial slower growth rate (0-2y) By year 2 the absolute growth rate of 0.07mmjday is equivalent to the Pacific sardine a temperate coastal pelagic. This is most striking considering the 10C temperature difference between the cold temperate and polar environments. Rather than being perceived as sluggish, slow growing fishes that occupy a midwater niche, it would perhaps be more accurate to think of

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Figure 22. Growth rate estimates for cold temperate and polar species. 2 epipelagic species, Sardinops caerulea (Lasker, 1970) and Engraulis mordax (Collins and Spratt, 1969); 2 cold temperate myctophids Stenobrachius leucopsarus (Smoker and Pearcy, 1970) and Benthosema glaciale (Gjsaeter, 1978), and the polar myctophid species, Electrona antarctica (present study)

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220 200 e18o 5160 .t: .... C) 140 c: (I) 120 -c I.. ca 100 -c c: 80 cu .... tJ) 60 40 20 0 0 S. caerulea E. mordax E. antarctica S leuco.P._ sarus ... 1 2 3 4 s s 1 8 g Estimated age (years) 98

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99 mesopelagic species as the sardines and anchovies of the open sea. The vertically migrating foraging strategy allows the myctophids a continual cloak of darkness to shield them from visual predation, while allowing them to maintain a zooplanktivorous feeding habit similar to that of the engraulids and clupeids.

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100 LITERATURE CITED Ainley, D.G., W.R. Fraser, c.w. Sullivan, J.J. Torres, T.L. Hopkins and W.O. Smith. 1986. Antarctic mesopelagic micronekton: evidence from seabirds that pack ice affects community structure. Science 232:847-849. Andriashev, A.P. 1965. A general review of the Antarctic fish fauna, pp. 491-550. In: J. van Mieghem and P. van Oye (eds) Biogeography and ecology of Antarctica. Junk Publ., The Hague. Bagenal, T.B. (ed). 1974. The Ageing of Fish. Old Working, Surrey, England. 234 pp. Blacker, R.W. 1974. Recent advances in otolith studies, pp. 67-90. In: F.R.H. Jones (ed) Sea Fisheries Research. Wiley, New York, NY. Brett, J.R. and T.D.D. Groves. 1979 energetics, pp 279-352. In: W.S. Hoar, J.R. Brett (eds) Fish Physiology, Vol. Press, London-New York. Physiological D.J. Randall and VIII. Academic Brothers, E.B. 1979. Age and growth studies on tropical fishes, pp. 119-136. In: S.B. Saila and P.M. Roedel (eds) Stock Assessment for Tropical Small-scale Fisheries. International Centre for Marine Resources Development, University of Rhode Island, Kingston, RI. Brothers, E.B., C.P. Mathews and R. Lasker. 1976. Daily growth increments in otoliths from larval and adult fishes. Fish. Bull. 74:1-8. Campana, S.E. 1983. Calcium deposition and otolith check formation during periods of stress in coho salmon, Oncorhynchus kisutch. Comp. Biochem Physiol. 75A:215220. Campana, S.E. and J.D. Neilson. 1985. Microstructure of fish otoliths. Can. J. Fish. Aquat. Sci. 42:1014-1032. Childress, J.J., Taylor, 1980. Patterns of reproduction in some Southern California. S.M., Cailliet, G.M., Price, M.H. growth, energy utilization and meso-and bathypelagic fishes off Mar. Biol. 61:27-40.

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101 Clarke, T.A. 1978. Diel feeding patterns of 16 species of mesopelagic fishes from Hawaiian waters. Fish. Bull. u.s. 75:495-513. Collins, R.A. and J.D. Spratt. 1969. Age determination of Northern anchovies, Engraulis mordax, from otoliths. In: J.D. Messersmith ( ed) The Northern anchovy (Engraulis mordax) and its fishery 1965-1968, pp. 39-55. Sacramento, California Department of Fish and Game 1969. (Calif. Fish Game Fish Bull. No. 147). Degens, E.T., W.G. Deuser and R.L. Haedrich. 1969. structure and composition of fish otoliths. 2:105-113. Molecular Mar. Biol. Donnelly, J., J.J. Torres, T.L. Hopkins and T.M. Lancraft. 1990. Proximate composition of Antarctic mesopelagic fishes. Mar. Biol. 106:13-23. Efremenko, V.N. 1985. the Southern Ocean. Illustrated guide to fish larvae of Biomass Sci. Ser. No. 5, 74pp. Efremenko, V.N. 1987. Distribution of eggs and larvae of Myctophidae in the southern Atlantic. J. Ichthyol. 26:141160. Gartner, J.V. Jr. 1991. Life histories of three species of lanternfishes (Pisces: Myctophidae) from the eastern Gulf of Mexico. I. Morphologica l and microstructural analysis of sagittal otoliths. Mar. Biol. 111:11-20. Geen, G.H., J D. Neilson, and M. Bradford. 1985. Effects of pH on the early development and growth and otolith microstructure of chinook salmon, Oncorhynchus tshawytscha. Can. J. Zool. 63:22-27. Geffen, A.J. 1982. Otolith ring deposition in relation to growth rate in herring (Clupea harengus) and turbot (Scophthalmus maximus) larvae. Mar. Biol. 71:317-326. Giragosov. B. Ye. and Ovcharov. 1992. Age and growth of the lantern fish Myctophum nitidulum (Myctophidae) from the tropical Atlantic. J Ichthyol. 32:34-42. Gj0saeter, H. 1987 Primary growth increments in otoliths 6 six tropical myctophid species. Biol. Oceano. 4: 3 59-382. Gjsaeter, J. 1978. Resource study of mesopelagic fish. PhD Thesis, University of Bergen, Bergen. 203pp.

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102 Gj0saeter, J. and K. Kawaguchi. 1980. A review of the world resources of mesopelagic fish. FAO Fish. Tech. Pap., Vol. 193, 151 pp. Haake, P.W., C.A. Wilson and J.M. Dean. 1982. A technique for the examination of otoliths by SEM with application to larval fishes, pp. 12-15. In: C.F. Bryan, J.V. Conner and F.M. Truesdale (eds) Proceedings of the Fifth Annual Larval Fish Conference. Hempel, I., G. Hubold, B. Kaczmaruk, R. Keller and R. Weigmann-Haass. 1983. Distribution of some groups of zooplankton in the inner Weddell Sea in summer 1979/80. First results of the 1979/80 "Polarsirkel" Expedition. Rep. Polar Res. No. 9, 36pp. Hopkins, T.L. 1985. Aspects of the trophic ecology of the mesopelagic fish Lampanyctus alatus (family Myctophidae) in the Eastern Gulf of Mexico. Biol. Oceano. 3:285-313. Hopkins, T.L. and R.C. Baird. 1985. Food web of an Antarctic midwater ecosystem. Mar. Biol. 89:197-212. Hopkins, T.L. and J.J. Torres. 1989. Midwater food web in the vicinity of a marginal ice zone in the western Weddell Sea. Deep-Sea Res. 36:543-560. Hopkins, T.L., D.G. Ainley, J.J. Torres and T.M. Lancraft. 1993. Trophic structure in open waters of the marginal ice zone in the Scotia-Weddell confluence region during spring (1983). Polar Biol. 13:389-397. Hulley, P.A. 1981. Results of research cruises of FRV 'Walther Herwig' to South America. LVIII. Family Myctophidae (Osteichthyes, Myctophiformes). Arch. Fisch Wiss. 31:1-300. Kock, K.H. 1987. Marine consumers: fish and squid. Environment International 13 :37-45. Kozlov, A.N. and M.I. Tarverdiyeva. 1989. Feeding of different species of Myctophidae in different parts of the Southern Ocean. J. Ichthyol. 29:160-167. Lancraft, T.M., T.L. Hopkins and J.J. Torres. 1988. Aspects of the ecology of the mesopelagic fish Gonostoma elongatum (Gonostomatidae, Stomiiformes) in the eastern Gulf of Mexico. Mar. Ecol. Prog. Ser. 49:27-40. Lancraft, T.M., J.J. Torres and T.L. Hopkins. 1989. Micronekton and macrozooplankton in the open waters near Antarctic ice edge zones (AMERIEZ 1983 and 1986). Polar Biol. 9:225-233.

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103 Lancraft, T.M., T.L. Hopkins, J.J. Torres and J. Donnelly. 1991. Oceanic micronektonicjmacrozooplanktonic community structure and feeding in ice covered Antarctic waters during the winter (AMERIEZ 1988). Polar Biol. 11:157-167. Lasker, R. 1970. Utilization of zooplankton energy by a Pacific sardine population in the California current. In: J.H. Steele (ed) Marine food chains, pp. 265-284. Oliver and Boyd, Edinburgh. Linkowski, T.B. 1985. Population biology of the myctophid fish Gymnoscopelus nicholsi (Gillbert, 1911) from the western South Atlantic. J. Fish Biol. 27:683-689. Linkowski, T. B. 1987. Age and growth of four species of Electrona (Teleostei, Myctophidae). Proc. V Congr. europ. Ichthyol., Stockholm 1985, pp.435-442. Linkowski, T. B. 1991. Otolith microstructure and growth patterns during the early life history of lanternfishes (family Myctophidae). Can. J. Zool. 69:1777-1792. Marshall, S.L. and s.s. Parker. 1982. Pattern identification in the microstructure of sockeye salmon (Oncorhynchus nerka) otoliths. Can. J. Fish. Aquat. Sci. 39:542-547. McGinnis, R.F. 1977. Evolution within pelagic ecosystems: Aspects of the distribution and evolution of the family Myctophidae, pp. 547-556. In: G G Llano (ed) Adaptations Within Antarctic Ecosystems, Proc. of SCAR on Ant. Biol. Smithsonian, Washington, D.C. McGinnis, R. F. 1982. Biogeography of lanternfisheE (Myctophidae) south of 30S. In: Biology of the Antarctic Seas XII. Washington, D C American Geophysical Unior (Antarctic Res. Series 35), 110pp. Methot, R.D. Jr. 1981. Spatial covariation of daily growtt rates of larval northern anchovy, Engraulis mordax, anc northern lampfish, Stenobrachius leucopsarus. Rapp. P.-v. Reun. Cons. Int. Explor. Mer. 178:424-431. Miya, M., M. Okiyama, and T. Nemoto. 1986. Midwater fishes o1 the Southern Ocean South of Australia (extended abstract). Mem. Natl Inst. Polar Res., Spec. Issue 40:323-324. Mugiya, V. and N. Watabe. 1977. Studies on fish scalE formation and resorption. II. Effect of estradiol 01 calcium homeostasis and skeletal tissue resorption in th goldfish, carassius auratus, and the killifish, Fundulu: heteroclitus. Comp. Biochem. Physiol. 57A:197-202.

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104 Mugiya, V., N. Watabe, J. Yamada, Dunkelberger, and M. Shimuzu. 1981. otolith formation in the goldfish, Comp Biochem. Physiol. 68A:659-662. J. M. Dean, D. G. Diurnal rhythm in Carassius auratus. North, A.W., M.G. White and M.S. Burchett. 1980. Age determination of Antarctic fish. Cybium 3:7-11. Ozawa T. and G.C. Pefiaflor. 1990. Otolith microstructure and early ontogeny of a myctophid species Benthosema pterotum. Nippon Suisan Gakkaishi 56:1987-1995. Pannella, G. 1971. Fish otoliths: daily growth layers and periodical patterns. Science 173:1124-1127. Pannella, G. 1974. Otolith growth patterns: an aid in age determination in temperate and tropical fishes, pp. 28-39. In: T.B. Bagenal (ed) The Ageing of Fish. Old Working, Surrey, England. Pannella, G. 1980. Growth patterns in fish sagittae, pp. 519-560. In: D.C. Rhoads and R.A. Lutz (eds) Skeletal Growth of Aquatic Organisms: Biological Records of Environmental Change. Plenum Press, N ew York, NY. Popper, A. N. and S. Coombs. 1980. Auditory mechanisms in teleost fishes. Amer Sci. 68:429-440. Prince, E.D., D.W. Lee, J.R. Zweifel and E.B. Brothers. 1991. Estimating age and growth of young Atlantic blue marlin, Makaira nigricans from otolith microstructure. Fish. Bull. 89:441-459. Radtke, R.L. 1984. Information incorporated in Antarctic fish otoliths. Proc. V Congr. europ. Ichthyol., Stockholm 1985, pp. 421-425. Radtke, R L and T.E. Targett. 1984. Rhythmic structural and chemical patterns in otoliths of the Antarctic fish Notothenia larseni: their application to age determination. Polar Biol. 3:203-210. Radtke, R.L., T.E. Targett, A. Kellermann, J.L. Bell and K.T. Hill. 1989. Antarctic fish growth: profile of Trematomus newnesi. Mar. Ecol. Prog. Ser. 57:103-117. Radtke, R.L. and T.F. Hourigan. 1990. Age and growth of the Antarctic f i s h Nototheniops nudifrons. Fish. Bull. 88:557-571. Reinhardt, S.B. and E.S. Van Vleet. 1986. Lipid compositior of twenty-two species of Antarctic midwater zooplanktor and fish. Mar. Biol. 91:149-159.

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107 APPENDICES

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108 APPENDIX 1. EXPANDED METHODOLOGY The following is a more detailed account of the results from application of various experimental methodologies attempted during the course of this study. 1) The larval zone (LZ and AP) was best enumerated from whole otolith preparations since frontal sectioning greatly compressed this region. This lateral compression made distinctions between daily and subdaily (finer) increments difficult further complicating enumeration. No etching was required to enumerate increments within this region. 2) In order to view microincrements using SEM, two etching techniques were attempted a physical process, reverse polarization and a chemical process. using reverse polarization were The results of etching inconclusive; this new application requires further modification, which precluded a detailed investigation during this study. Preliminary data, however, do provide promising results warranting further investigation. Two of the four sections processed using this technique did provide relief patterns visible with SEM. Follow-up applications will employ this technique using sagittae from a more suitable species, having a less complex morphology and which has been previously aged using daily increments. Chemical etching was attempted using solutions of

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109 APPENDIX 1. (Continued) EDTA and HCl. Optimal 3-dimensional relief was obtained using a 1% solution of HCl (pH 3 0) applied for 1-3 minutes. Applications of EDTA rendered only marginal results, yielding a 30% success rate. 3) Attempts to enumerate the PMZ from acetate replicates were unsuccessful. Results were inconsistent and complicated by extensive artifacts. Replicas attracted lots of debris and increment impressions were often unclear and smeared. The limitations of resolution using light microscopy (ca. 2 microns) may have contributed to the inconsistencies of this technique. Although microincrements were visible in several replicas the difficulties in interpretation deemed this technique unreliable. As outlined in Campana and Neilson (1985), microstructural detail can be lost during creation of impressions and visual artifacts can be introduced during subsequent examination with light microscopy. 4) The more laborious technique of SEM was utilized to enumerate the PMZ increments. Samples were prepared for SEM following the methods previously described (refer to Methods). Although SEM was the established protocol, successful preparations were fortuitous, as 33 of 60 attempts were successful.

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110 APPENDIX 2. INCREMENT COUNTS WITHIN THE LARVAL ZONE (LZ) Mean values recorded for each reader are the average of three to six counts. Overall mean values are the average of the three independent readers counts. The total mean value is the total average for all counts. A. Unsexed STANDARD LENGTH 23 mm 25 mm 27 mm 32 mm 33 mm 34 mm 36 mm 44 mm 44 mm 45 mm 49 mm 69 mm TOTALS READER 1 MEAN 28 35 31 36 36 37 34 40 48 47 47 30 12 READER 2 MEAN 27 36 30 32 35 38 35 39 46 46 46 29 12 READER 3 MEAN 27 36 31 36 36 34 48 7 OVERALL MEAN 27 36 31 36 36 37 34 39 47 46 46 29 37

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111 APPENDIX 2. (Continued) B. Females STANDARD READER 1 READER 2 READER 3 OVERALL LENGTH MEAN MEAN MEAN MEAN 43 mm 37 37 37 44 mm 44 43 43 45 mm 36 37 36 48 mm 47 49 48 49 mm 47 46 46 50 mm 35 35 35 51 mm 35 35 35 54 mm 35 35 35 56 mm 35 34 33 34 57 mm 43 43 43 61 mm 46 47 46 62 mm 46 44 45 64 mm 44 43 43 68 mm 37 36 36 71 mm 3 0 29 30 3 0 72 mm 32 30 31 31 74 mm 29 30 29 76 mm 39 38 38 38 77 mm 37 39 38 38 80 mm 40 40 40 81 mm 43 41 42

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112 APPENDIX 2. (Continued) B. Females (continued) STANDARD READER 1 READER 2 READER 3 OVERALL LENGTH MEAN MEAN MEAN MEAN 82 rnrn 34 34 34 83 rnrn 38 38 38 84 rnrn 37 39 38 90 rnrn 36 36 36 90 rnrn 45 44 44 92 rnrn 3 1 31 32 31 92 rnrn 37 38 38 38 97 rnrn 3 7 33 37 37 98 rnrn 40 40 40 103 rnrn 39 38 37 38 TOTALS 3 1 28 13 38

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113 APPENDIX 2. (Continued) c. Males STANDARD READER 1 READER 2 READER 3 OVERALL LENGTH MEAN MEAN MEAN MEAN 37 mm 31 30 30 40 mm 46 46 46 45 mm 44 42 43 45 mm 35 35 35 53 mm 30 31 29 30 56 mm 30 29 31 30 60 mm 35 32 35 35 60 mm 45 44 45 45 60 mm 49 47 48 65 mm 28 28 28 68 mm 44 45 43 44 69 mm 44 44 44 75 mm 38 39 38 76 mm 30 30 30 78 mm 38 37 38 38 81 mm 39 37 38 83 mm 36 34 35 TOTALS 17 17 6 37

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114 APPENDIX 3. INCREMENT COUNTS WITHIN THE POSTMETAMORPHIC ZONE (PMZ) PMZ counts are from scanning electron microscopy (SEM) photomontages of 3 2 sag i ttae. SL : standard length, U: unsexed, F: female, M: male, TR: sagitta total radius, LZR: sagitta larval zone radius, COUNT RANGE: range of all PMZ increment counts, MEAN COUNT: mean of all PMZ counts, LZ COUNT: larval zone increment count, TOTAL AGE: sum of mean PMZ count and LZ count. SL SEX (rom) 32 u 33 u 37 M 40 M 44 F 45 F 45 M 49 F 50 F 51 F 53 M 57 F 60 M 61 F 62 F 64 F 68 F TR (rom) 0.725 0.675 0.725 0.862 0.850 0.850 0.825 1.125 0.975 1.125 1.075 1.175 1. 275 1. 200 1.175 1. 225 1. 325 LZR (rom) 0.144 0.120 0.135 0.150 0.129 0.138 0.135 0.138 0.165 0.156 0.135 0.135 0.135 0.135 0.147 0.132 0 .135 COUNT MEAN LZ TOTAL RANGE COUNT COUNT AGE 296-314 303 36 339 270-295 280 36 316 382-426 419 30 449 351-366 357 46 403 480-501 492 43 535 496-540 527 36 563 503-549 528 43 571 570-614 583 46 629 587-632 610 35 645 609-664 654 35 689 629-662 647 30 677 708-751 736 43 779 775-836 800 48 848 780-808 793 46 839 788-838 805 45 850 829-860 843 43 886 871-912 904 36 940

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115 A P PENDIX 3 (Continued) SL SEX TR LZR COUNT MEAN LZ TOTAL (mm) (mm) (mm) RANGE COUNT COUNT AGE 68 M 1. 350 0 .126 947-969 954 44 998 69 M 1. 375 0 .129 985-1046 1017 44 1061 71 F 1. 350 0.135 946-986 966 30 996 75 M 1. 475 0 .120 1123-1187 1156 38 1194 76 F 1. 300 0 .150 978-1029 1009 38 1047 77 F 1. 375 0 .120 1033-1115 1055 38 1093 78 M 1. 550 0 .138 1094-1196 1144 38 1182 80 F 1. 375 0.120 1010-1063 1040 40 1 080 81 M 1. 500 0.138 1164-1202 1191 38 1229 83 M 1. 625 0 .120 1177-1249 1219 35 1254 83 F 1.550 0.150 1040-1092 1067 38 1105 84 F 1.650 0.135 1120-1153 1137 38 1175 92 F 1. 675 0.120 1158-1246 1180 31 1221 97 F 1. 750 0.150 1202-1263 1249 37 1286 98 F 1. 850 0.120 1184-1296 1255 40 1295 103 F 1 .790 0 .135 1236-1338 1317 38 1355


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