Validation of the otoliths for age determination in Barbus sclateri (Günth.)

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<ul><li><p>Ecology of Freshwater Fish 2001: 10: 122126 Copyright C Munksgaard 2001Printed in Denmark All rights reserved</p><p>ISSN 0906-6691</p><p>Short communication</p><p>Validation of the otoliths for agedetermination in Barbus sclateri (Gnth.)</p><p>Escot C, Granado-Lorencio C. Validation of the otoliths for age determi- C. Escot1, C. Granado-Lorencio2nation in Barbus sclateri (Gnth.). 1Aquatic Ecology Station, EMASESA, Sevilla,Ecology of Freshwater Fish 2001: 10: 122126. C Munksgaard, 2001 2Department of Plant Biology and Ecology,</p><p>Faculty of Biology, University of Sevilla, SpainAbstract Otoliths were used to explore the timing of annulus formationin Barbus sclateri. Lapillus exhibited opaque and translucent zoneswhose combination was formed once a year. Opaque zones appearedformed from July and January, and the translucent ones from February to Key words: otolith; age determination; Barbus</p><p>sclateriJune. We describe a procedure to assess age by using the number of an-nulii, the condition of the otoliths edge and the capture date. The use C. Granado-Lorencio, Department of Plantof otoliths for age determination in this cyprinid endemic to the Iberian Biology and Ecology, Faculty of Biology,</p><p>University of Sevilla, Apdo. 1095, SevillaPeninsula provides a useful alternative to the traditional methods of scaleE-41080, Spain; e-mail: granado/cica.esreading.Accepted for publication January 5, 2001</p><p>Un resumen en espanol se incluye detras del texto principal de este artculo.</p><p>Introduction</p><p>Studies on the age and growth of cyprinids en-demic to the Iberian Peninsula have focused on theinterpretation of fish vertebrae and scales. Whenthe first vertebrae is used, only specimens up to 8years have been reported due to imprecise determi-nation of rings in older individuals (Lucena &amp; Ca-macho 1978; Lucena et al. 1979). Moreover, Her-rera (1991) and Rodrguez-Ruiz (1992) reporteddifficulties in determining the location of the firstring in scales of older specimens due to the occur-rence of false marks.</p><p>Overall, otoliths have been considered themost accurate bone for age determination inslow-growing species (Beamish &amp; McFarlane1987), but its use to age cyprinids in uncommon.The objective of this study was therefore to vali-date otoliths for aging Barbus sclateri Gnther1868, one of most abundant members of streamfish assemblages in the southern Iberian Penin-sula. In particular, we attempt to detect annualformations in otoliths to meet the assumptionsestablished by Francis et al. (1992) and Francis(1995): 1) the existence of periodical marks inthe selected structure; 2) the ability to identifythese marks correctly (including discriminationfrom false marks); and 3) the ability to convertthe number of marks into age.</p><p>122</p><p>Material and methods</p><p>Otoliths of 340 specimens of B. sclateri (60315mm, standard length) were collected monthly withelectrofishing during a year in Rio Guadalete(southwestern Spain) and in Rio Bocaleones, amajor tributary. In contrast to the former stream,the latter is fed by springs and exhibits a ratherhomogeneous stream flow and thermal regimethroughout the year (Prenda 1993). The recentconstruction of a dam upstream the mouth of RioBocaleones into Rio Guadalete prevented mixingof the two fish populations during the last 3 years.</p><p>Otoliths were obtained from fresh fish just aftercollection or from individuals frozen for a maxi-mum of 2 hours after collection. The otoliths werecleaned in a 10% sodium hypochloride solution(bleach), and rinsed with distilled water. Then theywere immersed in 95% ethanol and stored dry.Based on previous assessments of these otoliths(Escot &amp; Granado-Lorencio, 1998), the right-sidelapillus was placed into water and observed with amicroscope at 40 using reflected light on a blackbackground. It was not necessary to clear thembecause these otoliths had clearly defined opaquezones alternating with translucent zones, and theoperation of reflected light made the opaque zoneappear white and luminous, and the translucentzone appeared dark.</p></li><li><p>Otoliths reading in Barbus sclateri</p><p>Fig. 1. A view of the whole lapillus of B. sclateri under magnify-ing glass, submerged into water on a black background withreflected light. The larger otolith above is from an 11 year oldfemale, 260 mm total length, captured in Rio Bocaleones inNovember (bar1 mm). The smaller otolith below is from a 6year old male, 92 mm total length, also captured Rio Bocaleon-es in January (bar0,5 mm). The black arrows indicate eachannulus</p><p>The distal portion of each translucent zone wasconsidered the annullus. The term annullus is as-sociated with the translucent zone that is formedonce a year in calcificed structures of fishes grow-ing in temperate climates (Casselman 1987). Theareas of complete growth for each year were repre-sented by the combination of one opaque zone anda subsequent translucent zone. The number of an-nuli of the whole lapillus was counted along thelongest antero-posterior axis on its dorsal face(anti-sulcal).</p><p>We applied the marginal increase analysismethod (Baillon 1992) to validate age. This tech-nique encompasses qualitative and quantitative ex-amination of the otolith edge over the growthperiod. The percentage of the otolith with opaqueor translucent zones on the edge was estimated(Ross &amp; Stevens 1995; Thompson &amp; Beckam 1995;Yosef &amp; Casselman 1995); the width of the twomost external zones of growth were measured(each opaque zone of the last annulus), and therelative width of the marginal increment (MI) wascalculated as the ratio of the width of the actualgrowth zone to the width of the previous yearsgrowth zone (Maceina &amp; Betsill 1987; Gooley1992; see also Kalish et al. 1995 for the glossaryof terms).</p><p>123</p><p>Results and discussion</p><p>Opaque and translucent zones in the whole lapilluswere clearly visible (Fig. 1). In several otoliths, thetranslucent zones were cleared to detect the mainaxis that became diffused when getting nearer thesuperior or inferior part of the otolith. Translucentzones were observed in the edge of the otolithsthroughout the year (Fig. 2a), but the proportionof otoliths with a translucent zone showed an un-imodal annual cycle. Over 80% of the otolithsshowed a marginal translucent zone in April andMay. More than 80% of the otoliths from Rio Gu-adalete exhibited a translucent edge in April, andthose from Rio Bocaleones exhibited a translucentedge between March and June (Fig. 2b, c).</p><p>Fig. 2. Plot of percentage of otoliths with translucent edges(bars) and of the mean marginal increments (dark squares, withstandard errors) during the study period. A. Individuals of B.sclateri from both streams pooled. B. Individuals from Rio Gu-adalete and C. Individuals from Rio Bocaleones</p></li><li><p>Escot &amp; Granado-Lorencio</p><p>Table 1. Analysis of the variance (anova) for the effects of capture time(month) and place (river) on the marginal increment (MI) of the otoliths of B.sclateri during the study period. The Bonferroni comparisons were used todetermine differences between adjacent means.</p><p>Source of Sum of Meanvariation squares df squares F P</p><p>Month 3.76 10 0.38 6.58 ,0.0001Place 0.09 1 0.09 1.56 0.212Month*Place 0.81 10 0.08 1.41 0.174Error 16.50 289 0.06</p><p>FebMarAprMay,JunJulAug,SepOctNovDec</p><p>Fig. 3. Monthly variations of the mean marginal increment forthe age-groups indicated in (a) Rios Guadalete and (b) Boca-leones (b)</p><p>The time of capture influenced significantly in-fluenced the amount of opaque material at theedge of the otolith (ANOVA, P,0.0001), but noenvironmental influence was significant (Table 1)(Fig. 2a, b, c). There were three differential stagesof otolith growth throughout the year. The otolithgrowth appeared at a minimum between Februaryto May and increased significantly between Juneand September.</p><p>Given that there was a clear pattern in theopaque zones growth for each river dataset, wefurther tested whether the same pattern occurredin all age-classes. We selected age-groups so thatwe could compare the greatest number of ages andyet have an adequate sample size. These were 24,</p><p>124</p><p>58 and 916 years in Rio Guadalete, and 24, 58, 912 and 1318 years in Rio Bocaleones. Asshown in Fig. 3 those patterns were consistentamong age-groups for both rivers.</p><p>Overall, the results confirmed the presence ofone opaque zone followed by a translucent zone inthe Barbels lapillus, and that the combination ofboth is formed once a year. A few false translucentzones were apparent, but they were easily detect-able because they did not occur in the areas aboveand below the nucleus. False zones were similar tothose of the annuli, but they were found in onlyone part of the structure, similar to the results ob-tained by Casselman (1987).</p><p>The formation of the annulus in calcified tissuesis complete when the translucent zone, whichforms once a year, is followed by opaque material(Casselman 1987). This corresponded to the mini-mum marginal growth values detected for our bar-bel population. Since, besides marginal growth, weincluded the periodical interpretation of the edgesappearance (opaque or translucent percentages),we were able to determine the timing of the forma-tion of the two zones: the opaque zone was formedin both populations from July to January and thetranslucent zone was formed from February toJune. Further observations on the reproduction ofB. sclateri in these streams suggest that repro-duction extends from February to June (Lucena &amp;Camacho 1978; Herrera et al. 1988; Herrera 1991;Rodrguez-Ruiz 1992).</p><p>Francis (1995) considered the interpretation ofthe edge as a major factor in estimating age, yet itis important to distinguish between the time of theyear when the zone is formed and the time whenit becomes visible. We found that the otoliths showvery fine translucent edges during the period whenopaque material in the edge most frequently ap-pears. This event has being termed pseudo-hyalineor pseudo-translucent edge (North 1988) andwould be the time in the otoliths growth whenthere is no difference between the opaque and thetranslucent zones. Also, Mina (1968) identified thisproblem of visual identification of a zone: ...wecan only observe that a new zone has been laid,when the change in the deposition of calcium hasbeen enough to produce a change in the opticaldensity of the material and therefore can be easilydetected... (see also Lecomte-Finiger 1992). Thisphenomenon can be further complicated whenworking with whole or sectioned otoliths where thezones correspond to three-dimensional layers, incontrast to fine sections in which the zones aretwo-dimensional structures (Francis 1992).</p><p>From July to January (the time of greater fre-quency of opaque edges), our observations of trans-lucent zones were probably misinterpreted, and this</p></li><li><p>Otoliths reading in Barbus sclateri</p><p>resulted from difficulty to distinguish betweentranslucent and the pseudo-translucent zones. Be-cause during this period there were no apparentgrowth in this pseudo-translucent edge, and therewas growth in the opaque zone, the formation of thelatter may have taken place before winter in Rio Gu-adalete and before the end of winter and the onsetof spring in Rio Bocaleones, and in the two rivers,the translucent zone was formed before the summer.</p><p>Once the rings were counted (annulli) they wereconverted into age. This last step is referred to byFrancis et al. (1992) as the interpretation of theedge. This implies a decision on the moment (ordate) when the most external ring counted wasformed. The conversion of the number of ringsinto age implies considerations on the relationshipbetween this date and two previous dates, i.e., timeof capture and birthdate (Francis 1995). For thispurpose, we took January 1 as the birth date forthe two fish stocks, which may not necessarily co-incide with the date of natural birth (Willliams &amp;Bedford 1974). Consequently, the precise age ofeach individual fish was determined taking the fol-lowing criteria into account: 1) If the otolith hadan opaque edge, the age was equal to the numberof annulli, regardless of the date of capture. 2) Ifthe otolith had a translucent edge, estimating theage depended on the date of capture as follows: iffish came from the July to December, the age wasequal to the number of annulli with a posterioropaque zone, and if the fish came from January toJune, age was equal to the number of annulli witha posterior opaque zone plus one.</p><p>In conclusion, if the condition of the edge of theotolith is taken into consideration, otolith readingprovides a useful alternative to other methods pre-viously applied for age determination in B. slcateriand probably in other cyprinids endemic to the Ib-erian Peninsula.</p><p>Resumen</p><p>1. Utilizamos otolitos para determinar el momento de la foma-cion de anillos anuales en Barbus sclateri. Examenes microsco-picos detallados pusieron de manifiesto la ocurrencia de zonasopacas y traslucidas en el Lapillus cuya combinacion se produceuna vez al ano. Las zonas opacas aparecieron desde Julio hastaEnero mientras que las translucidas lo hicieron desde Febrerohasta Junio.2. Describimos un procedimiento para evaluar la edad utilizan-do el numero de anillos, la condicion del lmite del otolito y lafecha de captura. La utilizacion de otolitos para determinar laedad de este ciprnido endemico de la Pennsula Iberica ofertauna alternativa a los metodos mas tradicionales basados en lec-tura de escamas.</p><p>Acknowledgements</p><p>This research was supported by the Department of Plant Bi-ology and Ecology and by the Aquatic Ecology Station of Sevil-</p><p>125</p><p>la. We thank to all those who assisted during the successivestages of this study, especially Dr. D. Rodriguez, Dr. L. Encina,E. Mellado and Dr. J. Prenda.</p><p>References</p><p>Baillon, N. 1992. Otolithometrie: Revue et problemes. In: Bagli-nie`re. J.L., Castanet, J., Conand, F. &amp; Meunier, F.J. (Eds.).Tissues durs et age individuel des Vertebres. Colloques et Se-minaires ORSTOM-INRA, Paris, pp. 2152.</p><p>Beamish, R.J. &amp; McFarlane, G.A. 1987. Current trends in agedetermination methodology. In: Summerfelt, R.C. &amp; Hall,G.E., ed. Age and growth of fish. Iowa State University Press,pp. 1542.</p><p>Casselman, J.M. 1987. Determination of age and growth. In:Weatherley, A.H. &amp; H.S. Gill, ed. The biology of fish growth.London: Academic Press, pp. 209242.</p><p>Escot, C. &amp; Granado-Lorencio, C. 1998. Morphology of theotoliths of Barbus sclateri (Pisces: Cyprinidae). Journal ofZoology 246, 8994.</p><p>Francis, R.I.C.C. 1992. Ageing of adult Snapper (Pagrus aur-atus) from otolith annual ring counts: validation by taggingand oxytetracycline injection. Australian Journal of Marineand Freshwater Research 43: 10691089.</p><p>Francis, R.I.C.C. 1995. The analysis of otolith data a math-ematicians perspective (What, precisely, is your model?). In:Secor, D.H., Dean, J.M. &amp; Campana, S.E., ed. Recent devel-opments in fish otolith research. Charleston: University ofSouth Carolina Press, pp. 8195.</p><p>Francis, R.I.C.C., Paul, L.J. &amp; Mulligan, K.P. 1992. Ageingof adult snapper (Pagrus auratus) from otolith annual ringscounts: validation by tagging and oxytetracycline injection.Australian...</p></li></ul>