Validation of daily increment deposition in otoliths. Age and growth determination of Aphia minuta (Pisces: Gobiidae) from the northwest Mediterranean

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  • M. Iglesias E. B. Brothers B. Morales-Nin

    Validation of daily increment deposition in otoliths.Age and growth determination of Aphia minuta(Pisces: Gobiidae) from the northwest Mediterranean

    Received: 30 December 1996 /Accepted: 16 April 1997

    Abstract The transparent goby Aphia minuta (Risso,1810) is one of the main target species of the small-scalefishery o the Island of Majorca. Otolith microstructureand length-frequency analysis were used to study the ageand growth of this species during the 1982/1983 and1992/1993 fishing seasons. Daily periodicity of incre-ment formation was determined by experiments withmarked otoliths in individuals maintained in captivity.The length range of the catches during the 11 yr periodwas between 12 and 49 mm, with a main distribution(89%) between 24 and 40 mm. Otolith age-readings in-dicate that the population exploited in the commercialfishery consists of seven age-groups (2 to 8 mo old), witha very high proportion of individuals (95%) between 3and 6 mo old. Population growth-curves revealed nodierences between males and females. The growth pa-rameters for the whole population are: asymptotic length,L1 53:69mm; growth coefficient; K 2:23 yr1; the-oretical age at length zero, t0 0:005 yr. Those indi-viduals of A. minuta caught in Majorca during the winterperiod reached a maximum age of 7 or 8 mo.


    The transparent goby Aphia minuta is a pelagic neriticgoby that belongs to a monotypic genus. The species is

    common in the European Atlantic from Gibraltar to thecoasts of Norway and the Baltic Sea, and in the Medi-terranean, including the Black Sea and the Azov Sea(Tortonese 1975). This goby is the object of an impor-tant small-scale fishery during the winter months (De-cember to March/May) o the island of Majorca(Balearic islands) (arrowed in Fig. 1), on sand and mudbottoms between 15 and 40 m in depth (Iglesias andMartorell 1992; Iglesias et al. 1994). Annually, the catchcomprises 4:5% of the commercialised landings in thefish market of Palma (sale centre for catches from thewhole island), and is normally caught in company with asmall number of two related goby species, Pseudaphiaferreri (De Buen and Fage, 1908) and Cristallogobiuslinearis (von Duben, 1845).

    There are fisheries similar to that of Majorca in otherareas of the Mediterranean (Fig. 1), such as the south-eastern Spanish coast (Martinez-Bano et al. 1990, 1993),northern Tyrrhenian (Serena et al. 1990), centralAdriatic (Froglia and Gramitto 1989) and southern Ad-riatic (Rizzoli 1983; Ungaro et al. 1994).

    Aphia minuta is a small species (not >60mm in totallength in the Mediterranean Sea) that gathers in shoalsclose to the bottom to spawn, after which it dies. Itprobably never lives longer than one year (De Buen1931). Little additional data are available on its lifehistory, and the biology of A. minuta is not very wellknown. Reproduction is from December to March in thewestern Mediterranean, in May in the Adriatic, fromMay to July in the Black Sea, and from July to August inthe Atlantic (Norway) (Fischer et al. 1987). It feeds onzooplankton, principally copepods and cirripede andmysid larvae (Miller 1986). The only available data onage and growth come from a preliminary study whichanalysed size frequencies and growth increments in theotoliths (Iglesias and Morales-Nin 1992).

    In recent years, numerous studies have demonstratedthat the examination of otolith microstructure andenumeration of daily growth increments (DGI) allow theage of individual fish to be determined with great pre-cision. This technique is of greatest use for obtaining

    Marine Biology (1997) 129: 279287 Springer-Verlag 1997

    Communicated by A. Rodrguez, Puerto Real

    M. Iglesias (&)Instituto Espanol de Oceanografa,Centro Oceanografico de Baleares, Apartado 291,E-07080 Palma de Mallorca, Spain

    E.B. BrothersEFS Consultants, 3 Sunset West, Ithaca,New York 14850, USA

    B. Morales-NinInstitut Mediterrani dEstudis Avancats (IMEDEA),CSIC/UIB, Campus Universitario,E-07071, Palma de Mallorca, Spain

  • information on the growth of larvae and juvenile fishesduring their first year of life (Campana and Neilson1985; Jones 1992). The short life of Aphia minuta makesDGI counts the ideal approach for obtaining informa-tion on its age and growth. However, in order to de-termine age based on growth-increment counts, it isnecessary to validate the periodicity of deposition(Beamish and McFarlane 1983; Geen 1992).

    The present study attempts to determine the size/agerelationship and the growth rate of Aphia minuta bymeans of otolith microstructure-analysis and length-frequency data. The periodicity of increment formationwas determined in experiments with marked otoliths ofindividuals maintained in captivity.

    Materials and methods

    Validation of daily increment formation in otoliths

    In order to confirm that the microstructural increments detected inthe otoliths of Aphia minuta (Risso, 1810) are formed daily, avalidation test was carried out in captivity by marking the otolithswith strontium chloride (SrCl2).

    In March 1994, 100 individuals of Aphia minuta (between 20and 40 mm in length) were captured at a depth of 30 m and a watertemperature of 13.5 C. On board ship, they were kept in a 50-litrecontainer without water renewal or aeration for 4 h. Upon landing,they were transported to an aquaculture station, where they wereplaced in a 250-litre tank, with flow-through seawater, constantaeration and an initial in situ temperature of 14.5 C. On the fol-lowing day, feeding with Artemia spp. (enriched with fatty acidsand vitamins) was commenced. After one week, the fish were ac-climatised to the environment and were responding favourably tothe diet. They were then marked by immersion in SrCl2 (a methodthat had been used with success for larvae, juveniles and adults ofother gobies: Brothers 1990). The procedure involved placing thefish in a static aquarium with a solution of 1.25 g SrCl2 per litre

    seawater for 24 h. After this time, the water was completely re-newed and the fish were returned to the initial holding conditions.This treatment produced bands of strontium-enriched material inthe otoliths that was detectable with a scanning electronic micro-scope (SEM), back-scattered electron image (BEI) and an energy-dispersive X-ray spectrometer (EDS) (Brothers 1990).

    Fifteen days after being marked, 15 individuals were sacrificed,and a similar number were subsequently sampled every 7 d. Thetotal number of marked and sacrificed individuals was 61; the studyended in mid-May. Between March and the end of the experimentin May, the water temperature increased from 14.5 to 20 C.

    In both the light microscopy (LM) and SEM preparations, aclear mark was observed at a distance from the otolith (sagitta)edge. Once SEM had confirmed that this mark corresponded withthe strontium mark in the otoliths of some individuals and that thethickness of presumptive DGIs was within the detection limit of theLM, the final counting of the DGIs from the Sr mark to the otolithborder was carried out with LM. The resolution of the mark byLM is due to the contrasting optical properties (greater refractiveindex) of strontium carbonate compared to the calcium carbonatepredominating in the otolith.


    From the 1982/1983 to the 1992/1993 fishing seasons (December toMarch/May), weekly and fortnightly (fishing season 1992/1993)length-frequency measurements were made on samples of Aphiaminuta obtained from landings of the commercial small-scale fish-ery at the central fish-auction wharf in Palma. Total fish length(TL) was measured to the nearest lowest millimetre (n 40681).

    Growth analysis was based on samples from the commercialcatches of Aphia minuta during the 1992/1993 fishing season (De-cember to March). During this period, eight fortnightly sampleswere taken. Total length was measured to the nearest millimetre for1007 individuals whose sizes ranged between 14 and 44 mm. Withineach sample, three individuals representative of each size intervalwere selected for biological measurements (n 394): wet weightwas measured to the nearest 0.01 g, sex was determined, and thesagittae were extracted.

    The otoliths were cleaned with a solution of KOH (5%), driedfor 24 h, mounted on slides for microscopy with Petropoxy, andpolished with a polishing rotating wheel and 0.3 lm alumina. The

    Fig. 1 Commercial fishingareas for Aphia minuta in Medi-terranean Sea (d), showingstudy site (arrowed )


  • sagittal sections thus obtained displayed a complete increment-counting path from the primordium to otolith margin. The sectionsfor SEM observation were decalcified to reveal the increment se-quence using 0.1 M EDTA (ethylenediamine tetraacetic acid)(Brothers 1990; Morales-Nin 1992).

    Age interpretation

    Increment reading was carried out under LM. Pending validation,growth increments were presumed to have been deposited daily andtotal counts could be equated to age in days. The microscope had ahigh-resolution television camera attached, and the image of theotolith was displayed on a closed-circuit television monitor. Theincrements were read on the monitor along the longest radius of theotolith, from the primordium to the margin. Two readings weremade for each otolith. The magnifications used for reading theDGIs were 630 and 1000, with oil immersion and plane-polarisedlight.

    The mean of the two readings (when counts did not dier by >5increments) was considered as the age in days for each individual.As it was not known when the formation of the increments began,the age obtained was approximate, and lacked a correction factorcorresponding to the period between hatching and initiation ofincrement deposition.

    Otolith dimensions (minimum and maximum diameters) weremeasured using an analysis-image system (MIP, Microm Espana)with an iterative measurement application.

    Growth calculation

    The lengthweight relationship was calculated for the 1992/1993fishing season both for males, females and indeterminates sepa-rately and for the population as a whole. For the entire 1985/1986to 1992/1993 study period, only the population relationship wasdetermined. The applied power regression equation was:

    Total weight (mg) a total lengthb (mm) : 1The allometric indexes (b) for the males, females and indeter-

    minates, and the possibility of isometry were examined by a Stu-dents t-test (Sokal and Rohlf 1981), using the expression:

    t b1 b2=Sb1b2 ; 2where Sb1b2 standard error of the dierence between the re-gression coecients.

    The growth parameters of the von Bertalany (1938) equa-tion were calculated for males and females and for the popula-tion as a whole by means of a statistical programme, Fishparm(Prager et al. 1987), based on the sizeage pairs of values de-termined by the DGI count in the otoliths and using the Mar-quadt algorithm for the iterative calculation of the parameters.Growth parameters based on length-frequency were also esti-mated using the Complete Elefan software package (Gayaniloet al. 1988) for the 1992/1993 fishing season and for all fishingseasons (1982/1983 to 1992/1993) combined.

    As L1 and K are correlated, the growth performance indexU 2 log L1 log K (Munro and Pauly 1983) was used to com-pare the growth parameters estimated by these two methods withthe growth parameters of other gobies recorded in the literature.

    Weight, W1 was calculated using the lengthweight relationshipparameters for the entire population and the L1 calculated bymeans of the DGI count:

    W1 a Lb1 : 3


    Lengthweight relationship

    Table 1 gives the parameters of the lengthweight rela-tionship for male, female and indeterminate Aphia mi-nuta alone, and for the whole population. Positiveallometric growth was observed in both sexes and for thewhole population. A Students t-test did not reveal anysignificant dierences between the allometric coecientin any of the size-ranges considered ( p < 0:05).

    Otolith characteristics

    The sagittae of Aphia minuta were of a concaveconvexform, disc-shaped for small-sized individuals and be-coming slightly oval in form with increasing size. In oursamples, minimum diameter (Dmin) ranged between 0.26and 0.82 mm, and maximum diameter (Dmax) was be-tween 0.31 and 1.16 mm. Relationship between thesediameters and total fish length was linear for each otolithdiameter, confirming a direct relation between oto-lith diameter and fish growth:

    Dmin 0:08806 0:02032 TL r2 0:89 ; 4aDmax 0:15738 0:02725 TL r2 0:87 : 4b

    LM observation revealed an elongated primordium(Fig. 2a), occasionally slightly expanded at the extrem-ities and surrounded by increments composed of cleardark (D) and light (L) zones similar to deposition layersof other fish species. The first increments formed aroundthe primordium were very fine and, in some cases, dif-ficult to resolve (arrowed in Fig. 2a). The thickness ofthe increments increased gradually with otolith growthbut with no sign of secondary nuclei or transition zones,

    Table 1 Aphia minuta. Esti-mated parameters a and b ofrelationship y a xb betweentotal length and total weight (r2

    determination coecient; nnumber of specimens)

    a b r2 (n) Range(mm)

    1992/1993Males (M) 0.000492 3.64 0.95 (182) 2344Females (F) 0.000529 3.62 0.96 (168) 2244M+F 0.000512 3.63 0.95 (350) 2244Indeterminates (I) 0.000633 3.54 0.92 (117) 1638Total (M+F+I) 0.000407 3.69 0.96 (467) 1644

    1985/1986 to 1993/1994Total 0.00071 3.55 0.93 (1965) 1645


  • and then diminished in the marginal zone of the largersized specimens (Fig. 2b).

    Those sections etched with EDTA and observed bySEM showed the microarchitecture of Aphia minutaotoliths to be characterised by small needle-shapedcrystals. This particular microstructure and the contin-uous addition of crystalline prisms perpendicular to theotolith surface made it dicult to obtain sections withdefined DGIs observable by SEM. The LM preparationsallowed a greater depth of field, and revealed the in-crements with greater clarity (Fig. 2b).

    DGI validation

    SEM observations demonstrated that we could clearlydistinguish the strontium-enriched band as a brighterconcentric mark in the sectioned otoliths of labelled fish.At greater magnification (LM: Fig. 2c), it was apparentthat the number of rings between the strontium markand the edge of the otolith coincided with the dayselapsed between labelling and subsequent sacrifice of theindividuals (Otolith F17, Table 2).

    A total of 18 otoliths (30% of those labelled) dis-played a clear strontium mark with well-defined incre-ments, allowing a DGI count between the mark and the

    otolith margin (Table 2). The relationship between thenumber of DGIs and the days elapsed (DE ) from la-belling was not statistically dierent from 1:1.

    DGI DE 0:13723 r2 0:99 : 5Thus, the increments formed in the otoliths of Aphia

    minuta were deposited daily. In the micrographs(Fig. 2d) a zone of slow otolith growth (fine increments)appears just before the strontium mark in the otolith.This zone consisted of approximately seven increments7 d, a period that corresponds to the interval be-twe...


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