Otoliths are acellular concretions of calcium carbonate (aragonite crystals) and other inorganic salts1–3 that develop in a protein matrix in the inner ear of osteichthyan species and are closely associated with the sensitive maculae of the labyrinthic compartments. Otoliths consist of 3 pairs of structures called sagittae, lapilli, and asterisci. Among the 3 types of otoliths, sagittae have the largest morphological variability in terms of visibly distinct opaque and translucent zones and are most frequently studied as a reliable indicator of age in fish.4–6 In addition to the determination of fish age via examination of otoliths, the relationship between fish length and otolith length has also been studied.7,8 The morphology of otoliths varies greatly among fish species. Otoliths are 3 times as dense as the rest of the skeleton and have essentially static and otic functions.9,10
Bogue (Boops boops) is an economically and scientifically important species of sparid fish in the Mediterranean region.11,12 This common sea bream is a demersal and semipelagic species living on all types of seabed (sand, mud, rock, and seagrass beds) and can be found in waters as deep as 350 m. The maximum recorded length of these fish is 40 cm, although sizes more typically range from 10 to 25 cm.13
Studies14–21 on the growth and reproductive biology of bogue have been performed on the Atlantic and Mediterranean coasts. This fish is a protoginous hermaphrodite that reaches sexual maturity in the Mediterranean Sea at 1 to 2 years of age.15,19,20 In the western Mediterranean Sea, spawning occurs between February and July, whereas in the Atlantic Ocean, it occurs between February and June.15,18–20 Bogue are omnivorous; juveniles are generally carnivores, and adults are primarily herbivores. In particular, bogue feed on benthic (crustaceans, mollusks, Annelida, and Sipuncula) and pelagic (Siphonophorae, copepods, and eggs) prey and algae, and Chlorophyta represent a large proportion of the diet.22
The objective of the study reported here was to validate the use of radiography to determine the length of paired sagittal otoliths in intact bogue cadavers. We also sought to determine the optimal radiographic view for assessment of sagittal otoliths by evaluating measurements on radiographs obtained via ventrodorsal as well as 30° right dorsal–left ventral and 30° left dorsal–right ventral oblique projections. Bogue were used in the study reported here because they were readily available and easily obtained.
Materials and Methods
Intact bogue (Boops boops) cadavers (n = 52 fresh samples) were obtained from commercial markets in Messina, Sicily. Specimens were selected within as broad a size range as possible. The fish were caught in the Tyrrhenian Sea from January to February 2010. Fish were weighed, and their standard length (measured from the tip of the snout to the caudal end of the last vertebra) was measured to the nearest millimeter by 1 investigator (RB).
Radiographic examination of bogue cadavers was performed by use of standard radiographic equipment used for small animal patients. Because the width of the examined region was < 10 cm, a grid was not needed. The radiographic beam was centered over the area of the inner ear, in the postorbital region behind the brain. A focal spot size of 50 × 50 mm was used. Radiographs were obtained with exposure settings of 40 kV and 4 mAs for fish ranging from 90 to 160 mm in length; settings of 40 kV and 6.5 mAs were used for fish with a length between 161 and 255 mm. All fish were examined in 3 radiographic views in the following order: ventrodorsal, 30° right dorsal–left ventral oblique, and 30° left dorsal–right ventral oblique.
For right dorsal–left ventral oblique and left dorsal–right ventral oblique projections, fish were positioned in left and right lateral recumbency, respectively, and 30° wedges of radiotransparent foam were used for vertebral column elevation. In ventrodorsal views, forceps or sandbags were used for positioning. After radiography was completed, the gill isthmus was manually torn and the head was reflected away from the gills and the rest of the body. Otoliths were removed, and 2 investigators (FM and RB) measured the length (rostrum-antirostrum) of sagittal otoliths (Figure 1) directly by use of a vernier caliper with a standard reading error of 1/20 mm = 0.05 mm. Measurements on radiographic images were also obtained with a vernier caliper. On ventrodorsal views, left and right sagittae were measured. In 30° right dorsal–left ventral oblique views, only the right sagittal otolith was measured, and in 30° left dorsal–right ventral oblique views, only the left sagittal otolith was measured. Contralateral otoliths could not be correctly measured in these views because of image magnification artifacts. For all fish, the 3 radiographic views were independently evaluated by the same investigators for measurement of sagittae on each radiograph. These individuals were blinded in regard to direct measurements of the otoliths. The mean of radiographic measurements obtained by the investigators was used in the subsequent analysis.
Photographs showing a stereomicroscopic view of the right sagittal otolith from a representative bogue (Boops boops) cadaver in a study to evaluate the use of radiography for determination of sagittal otolith length in 52 of these common sparid fish. The rostral aspect of the otolith is oriented toward the top in each image. 1 = Dorsoventral view. 2 = Lateral view. Ruler = 1 cm.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Photographs showing a stereomicroscopic view of the right sagittal otolith from a representative bogue (Boops boops) cadaver in a study to evaluate the use of radiography for determination of sagittal otolith length in 52 of these common sparid fish. The rostral aspect of the otolith is oriented toward the top in each image. 1 = Dorsoventral view. 2 = Lateral view. Ruler = 1 cm.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Photographs showing a stereomicroscopic view of the right sagittal otolith from a representative bogue (Boops boops) cadaver in a study to evaluate the use of radiography for determination of sagittal otolith length in 52 of these common sparid fish. The rostral aspect of the otolith is oriented toward the top in each image. 1 = Dorsoventral view. 2 = Lateral view. Ruler = 1 cm.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Statistical analysis—The assumption of normality of data was tested by use of a Shapiro-Wilk test. Sagittal otolith lengths measured directly were not normally distributed (W = 0.81; P < 0.01). The relationship between direct and radiographic measurements of sagittal otolith length was established according to the following equation: Y = a + bX, where Y is the length of a sagittal otolith measured on a radiograph, X is the directly measured length of the same otolith, and a is the intercept and b is the slope of a line in which a and b are estimated by use of the least squares method.23 This relationship was established between direct and radiographic measurements in ventrodorsal views and between direct and radiographic measurements for each oblique view. After logarithmic transformation, the relationship between sagittal otolith length and fish length (both measured directly) was also computed according to the equation Y = a + bX, where Y is the fish length and X is the sagittal otolith length.23 A Pearson correlation coefficient was calculated for each regression line. Analysis of covariance was used to compare the slopes (b values) of the regression lines obtained from ventrodorsal and oblique measurements of otoliths. Values of P < 0.05 were considered significant.
Results
Although some bones of the skull in bogue could not be clearly distinguished in radiographs, many major features (eg, cranial bones, cervical vertebrae, jaw, and operculum) were identified in the ventrodorsal and oblique views. Otoliths were the most prominent features of the skull in radiographs and served as clear reference points in the caudal aspect of the skull (Figure 2). Sagittal otoliths were identified as paired structures arranged on 2 parallel para-sagittal planes and were comma-shaped in ventrodorsal projections. In oblique projections, they were ovoid in shape and both were located on the same dorsal plane. Sagittae were subjectively larger and more dense than were lapilli, which were detected as paired ovoid otoliths rostral to the sagittae, and asterisci, which were identified caudal to the sagittae.
Ventrodorsal (A) and 30° right dorsal-left ventral oblique (B) radiographic views of the skull in representative bogue cadavers. In panel A, the radiopaque frame around the fish is created by forceps used to maintain the correct positioning. The 3 paired otoliths are easily detected on the images. 1 = Lapilli. 2 = Sagittae. 3 = Asterisci.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Ventrodorsal (A) and 30° right dorsal-left ventral oblique (B) radiographic views of the skull in representative bogue cadavers. In panel A, the radiopaque frame around the fish is created by forceps used to maintain the correct positioning. The 3 paired otoliths are easily detected on the images. 1 = Lapilli. 2 = Sagittae. 3 = Asterisci.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Ventrodorsal (A) and 30° right dorsal-left ventral oblique (B) radiographic views of the skull in representative bogue cadavers. In panel A, the radiopaque frame around the fish is created by forceps used to maintain the correct positioning. The 3 paired otoliths are easily detected on the images. 1 = Lapilli. 2 = Sagittae. 3 = Asterisci.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Scatterplots of ventrodorsal (A) and oblique (B) radiographic measurements of right and left sagittal otoliths and of standard fish length (measured from the tip of the snout to the caudal end of the last vertebra; C) versus directly measured sagittal otolith length in 52 bogue. In panel B, right sagittal otoliths were measured on 30° right dorsal–left ventral oblique views, and left sagittal otoliths were measured on 30° left dorsal–right ventral oblique views.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Scatterplots of ventrodorsal (A) and oblique (B) radiographic measurements of right and left sagittal otoliths and of standard fish length (measured from the tip of the snout to the caudal end of the last vertebra; C) versus directly measured sagittal otolith length in 52 bogue. In panel B, right sagittal otoliths were measured on 30° right dorsal–left ventral oblique views, and left sagittal otoliths were measured on 30° left dorsal–right ventral oblique views.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Scatterplots of ventrodorsal (A) and oblique (B) radiographic measurements of right and left sagittal otoliths and of standard fish length (measured from the tip of the snout to the caudal end of the last vertebra; C) versus directly measured sagittal otolith length in 52 bogue. In panel B, right sagittal otoliths were measured on 30° right dorsal–left ventral oblique views, and left sagittal otoliths were measured on 30° left dorsal–right ventral oblique views.
Citation: American Journal of Veterinary Research 73, 2; 10.2460/ajvr.73.2.233
Mean values were determined for left and right sagittal otoliths measured directly, measured in ventrodorsal radiographic projections, and measured in oblique radiographic projections. Standard length of the fish ranged from 90 to 255 mm, and weight ranged from 10.94 to 210.10 g. Correlations between the length of sagittal otoliths measured directly and that measured on radiographs were calculated, and data were summarized (Figure 3; Table 1).
Correlation coefficient (r), intercept (a), and slope (b) for the relationship between direct and radiographic measurements of sagittal otolith length in a study of 52 bogue (Boops boops) cadavers.
| Projection | Sagittal otolith | r | a | b | P value |
|---|---|---|---|---|---|
| Ventrodorsal | Right | 0.997 | 0.124 | 0.985 | < 0.001 |
| Left | 0.999 | −0.018 | 1.002 | < 0.001 | |
| Oblique | Right | 0.972 | 0.22 | 0.965 | < 0.001 |
| Left | 0.972 | 0.085 | 0.981 | < 0.001 |
Oblique measurements for right and left sagittal otoliths were performed on 30° right dorsal–left ventral oblique views and on 30° left dorsal–rightventral oblique views, respectively.
The median difference between sagittal otolith length measured directly and that measured on ventrodorsal radiographs for any fish was 0 mm (differences ranged from 0 to 0.4 mm); for oblique views, the median difference was 0.25 mm (differences ranged from 0.10 to 0.65 mm). Radiographic length of sagittal otoliths was significantly correlated with directly measured length (Table 1).
The slopes of the regression lines (b values) for ventrodorsal and oblique measurements of sagittal otoliths were not significantly (P = 0.45) different (Figure 3). Thus, both views were useful for determination of sagittal otolith length. Sagittal otolith length was also significantly correlated with standard fish length (right sagittal otolith, Y = 1.376X + 2.606 [r = 0.964]; left sagittal otolith, Y = 1.403X + 2.556 [r = 0.966]).
Discussion
It is widely known that otolith morphology varies with fish age, especially during early life stages. Otolith shape and dimension can also be related to geographic location, ocean depth, and chemical and physical qualities of the environment.24–26
The use of radiography to study otoliths in teleost fish has already been reported.27 To the authors' knowledge, 1 investigator has reported on the relationships between the length of fish and length of the left otolith sagittal otolith obtained from radiographic images (in lateral projection) or from direct measurements and found these to be significantly different.28 Results of the study reported here indicate that the measurement of sagittal otoliths on ventrodorsal and oblique radiographic images of fish is as accurate as direct measurement, and in contrast to findings of the previous study,28 radiographic length of sagittal otoliths was significantly correlated with their directly measured length. Use of a small radiographic beam focal spot minimizes the scatter typically associated with magnification in radiographic images. The use of sandbags or forceps to maintain correct positioning is acceptable if they do not obstruct the anatomic structures in ventrodorsal projections. Because of the different sizes of fish cadavers used in the present study, positioning was more difficult for oblique than for ventrodorsal projections. Once fish were properly positioned, otoliths were easily identifiable in radiographs.
Radiographic examination of sagittae is useful to determine the length of sagittal otoliths and, consequently, the fish length. With regard to ecological applications, the radiographic measurement of sagittal otolith length may be used for studies on the aquatic diet of organisms (fish, pinnipeds, and marine birds) because it allows for quick back-calculation to the size of prey.27,29
Although the results of statistical analysis indicated that the measurement of sagittal otoliths in the ventrodorsal and oblique projections were similarly useful for the determination of actual (direct) sagittal otolith length, these were subjectively more difficult to measure in oblique projections because of reflection of the matching contralateral otolith and because of image deformation. In the authors' opinions, the use of ventrodorsal radiographs for this purpose was simpler and more immediate.
Results of the present study indicate that radiographic examination is an accurate noninvasive procedure for measurement of sagittal otoliths in intact bogue cadavers and yields results comparable to those obtained via direct measurement. Ventrodorsal and oblique projections were similarly accurate for the determination of sagittal otolith length, but the authors were most confident in measurements obtained on ventrodorsal projections.
References
- 1.
Hildebrand M. Analysis of vertebrate structure. New York: John Wiley & Sons, 1988.
- 2.
Weichert CK, Presch W. Elementos de anatomia de los cordados. Mexico City: McGraw-Hill, 1981.
- 3.
Jobling M. Environmental biology of fishes. London: Chapman & Hall, 1995.
- 4.
Degens ET, Deuser WG, Haedrich RL. Molecular structure and composition of fish otoliths. Mar Biol 1969; 2:105–113.
- 5.
Campana SE. Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Mar Ecol 1999; 188:263–297.
- 6.
Popper AN, Coombs S. The morphology and evolution of the ear in Actinopterygian fishes. Am Zool 1982; 22:311–328.
- 7.
Secor DH, Dean JM. Somatic growth effects on the otolith-fish size relationship in young pond-reared striped bass, Morone sexatilis (Walbaum). Can J Fish Aquat Sci 1989; 46:113–121.
- 8.
Akyol O, Metin G, Unsal S. Relationship between otolith to fork lengths of sardine (Sardina pilchardus Walbaum, 1792) in the bay of Izimir (Aegean Sea), in Proceedings. Mediterranean Fisheries Cong 1997; 925–929.
- 9.
Grassé PP. L'oreille et ses anexes. In: Grassé, PP, ed. Traité de zoologie. Vol XIII. Paris, Masson & Cie Ltd, 1958;1063–1098.
- 10.
Ramcharitar J, Deng X, Ketten D, et al. Form and function in the unique inner ear of a teleost: the silver perch. J Comp Neurol 2004; 475:531–539.
- 11.
Bauchot, ML, Hureau JC. Sparidae. In: Whitehead PJP, Bauchot ML, Hureau JC, et al, eds. Fishes of the North-eastern Atlantic and the Mediterranean (FNAM). Paris: UNESCO Publishing, 1986;883–907.
- 12.
Lleonart J, Maynou F. Fish stock assessments in the Mediterranean: state of the art. Scientia Marina 2003; 67(suppl 1):37–49.
- 13.↑
Gordo LS. On the age and growth of the bogue, Boops boops (L.), from the Portuguese coast. Fish Manag Ecol 1996; 3:157–164.
- 14.
Abecasis D, Bentes L, Coelho R, et al. Ageing seabreams: a comparative study between scales and otoliths. Fisheries Res 2008; 89:37–48.
- 15.↑
Monteiro P, Bentes L, Coelho C, et al. Age and growth, mortality, reproduction and relative yield per recruit of the bogue, Boops boops Linné, 1758 (Sparidae), from the Algarve (south of Portugal) longline fishery. J Appl Ichthyol 2006; 22:345–352.
- 16.
Hernandez AV. Study on the age and growth of bogue (Boops boops L.) from the central Adriatic Sea. Cybium 1989; 13:281–288.
- 17.
Khemiri S, Gaamour A, Zylberberg L, et al. Age and growth of bogue, Boops boops, in Tunisian waters. Acta Adriat 2005; 46:159–175.
- 18.
Gordo LS. On the sexual maturity of the bogue (Boops boops) (Teleostei, Sparidae) from the Portuguese coast. Sci Mar 1995; 59:279–286.
- 19.
El-Agamy A, Zaki MI, Awad GS, et al. Reproductive biology of Boops boops (Family Sparidae) in the Mediterranean environment. Egypt J Aqaut Res 2004; 30:241–245.
- 20.
Bensahala Talet A, Belaouda D, Matoub L. Periode de ponte et taille a la premiere maturitè sexuelle de Boops boops (Linne, 1758) des Cotes Oranaise (Algerie). Rapp Comm Int Mer Medit 1990; 32:1.
- 21.
Relini G, Bertrand J, Zamboni A. Synthesis of the knowledge on bottom fishery resources in central Mediterranean (Italy and Corsica). Biol Mar Medit 1999; 6(suppl 1):382–386.
- 22.↑
Derbal F, Kara MH. Composition du régime alimentaire du bogue Boops boops (Sparidae) dans le golfe d'Annaba (Algérie). Cybium 2008; 32:325–333.
- 23.↑
Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. 3rd ed. New York: W. H. Freeman & Co, 1995;887.
- 24.
Kinagigil HT, Akyol O, Metin G, et al. A Systematic study on the otolith characters of Sparidae (Pisces) in the Bay of Izmir (Aegean Sea). Turk J Zool 2000; 357–364.
- 25.
Wilson RR Jr. Depth-related changes in sagitta morphology in six macrourid fishes of the Pacific and Atlantic oceans. Copeia 1985; 4:1011–1017.
- 26.
Campana SE, Neilson JD. Microstructure of fish otoliths. Can J Fish Aquat Sci 1985; 42:1014–1032.
- 27.↑
Ross RMH. Johnson JH, Adams CM. Use of fish otolith-length regressions to infer size of double-crested cormorant prey fish from recovered otoliths in Lake Ontario. Northeast Nat 2005; 12:133–140.
- 28.↑
Pedersen J. Comparison of vertebrae and otoliths measured directly and from radiographs. Fish Res 1997; 29:277–282.
- 29.
Tarkan AS, Gursoy Gaygusuz C, Gaygusuz Ö, et al. Use of bone and otolith measures for size-estimation of fish in predator-prey studies. Folia Zool 2007; 56:328–336.
