52 



ONTOGENY AND SYSTEMATICS OF FISHES-AHLSTROM SYMPOSIUM 





Fig. 22. Photomicrographs of otoconia in teleosts. (A) Bonefish, Albula vulpes. free otoconia. (B) Bonefish, otoconia embedded m the sagitta. 



General Methodology 



The otoliths (sagittae, lapilli, and asterisci) of larval fish are 

 usually the first calcified structures to appear in the development 

 of an individual. At least some of the otoliths are frequently 

 evident before hatching. Over the larval life, they vary in size 

 from a few to several hundred micra for different taxa and ages. 

 Because of their composition and small size (high surface to 

 volume ratio), larval otoliths are very sensitive to degradation, 

 decalcification, and dissolution in acidic solutions (McMahon 

 and Tash, 1979), and great care must be exercised in preserving 

 larval fish and otoliths. Improper handling results in rapid and 

 irreversible damage. Fresh larvae are best stored for later otolith 

 extraction in three ways: 1 ) frozen, 2) fixed and maintained in 

 strong ethanol solutions (preferably 95%), 3) dried (e.g., on glass 

 slides). The last technique is least preferred due to increased 

 difficulties in otolith removal and general damage to the larvae. 

 Removal from embryos and larvae involves microscopic dis- 

 section with fine needles. The use of crossed polarized filters is 

 sometimes helpful in locating the otoliths, although they are 

 generally clearly visible in the otocysts or otic capsules with 

 standard transmitted illumination. Dissection is best carried out 

 in water, and opaque larva can be cleared by brief exposure to 

 a weak KOH (1%) solution. Air dried otoliths should be trans- 

 ferred on the tips of oil wetted (immersion) needles, and for 

 light microscopy may be stored in oil on slides or permanently 

 mounted under coverslips with a neutral medium (non-acidic). 

 In the latter case, care must be taken to prevent the otoliths 

 from being cracked or crushed as the mounting medium shrinks 

 and pulls down the coverslip. In most cases larval otoliths are 

 small and thin enough to preclude a need for grinding. Light 

 microscopy is best applied to studies of internal structures, al- 

 though some external features can be viewed with either surface 

 microscopy or transmitted light and wide openings of the con- 

 denser diaphragm. Compound microscopes should have high 

 quality oil immersion optics (preferably to at least 1 ,000 x ) and 

 polarizing filters. For the latter, a single, rotatable field polarizer 

 helps in resolving internal structures, while an analyzing polar- 

 izer can be employed to locate the very small, but highly bire- 

 fringent otoliths on slides. A moderately high resolution (at least 

 500 lines) black and white video system is an additional, but 

 invaluable accessory. Such a system reduces eye fatigue, sim- 



plifies group viewing, measurement and photography, and most 

 importantly can substantially enhance image quality by elec- 

 tronic adjustment. It is also a necessary component in a variety 

 of automatic and semi-automatic image analysis systems. 



Scanning electron microscopy is most useful for high reso- 

 lution views of external structures, for examination of fine (< 1 

 fim) internal features, and for confirmation of suspected optical 

 artifacts. However the technique is also more expensive and 

 time consuming and may necessitate critical preparation. Whole, 

 cleaned and air-dried otoliths can be mounted and coated by 

 standard techniques. Internal views require embedding, grind- 

 ing, polishing and etching before stub mounting and coating. 

 The most recent important development in SEM preparation 

 is the use of etching solutions other than the initially preferred 

 HCl. Haake et al. (in press) summarize a technique for SEM 

 preparation of larval otoliths. 



Otolith Morphology and Early Ontogeny 



There are a number of papers which deal with the general 

 structure and composition (Hickling, 1931; Degens et al., 1969: 

 Blackler, 1974: Pannella, 1980), mechanism of growth (Irie, 

 1960: Dunkelburgeretal., 1980; Campana, 1983), and functions 

 of the otoliths and otolith organs (Popper and Coombs, 1 980a, b; 

 Piatt and Popper, 1981). This work has not specifically dealt 

 with larvae, however the gross morphology and processes should 

 be comparable with older fishes. 



The otic capsule or otocyst forms very early in the ontogeny 

 of fishes and is an obvious landmark in the head of newly 

 hatched larvae. The earliest evidence of the otoliths is one to 

 several small (usually less than 10 ixm) optically dense bodies, 

 referred to here as primordia. From their physical appearance 

 and etching properties, the primordia are assumed to be sub- 

 stantially composed of organic matrix (probably the fibroprotein 

 otolin), and are soon calcified and surrounded by an accreted 

 layer of calcium carbonate and matrix. There are distinct dif- 

 ferences between certain taxa, usually at the supraspecific level, 

 with regards to the morphology of the primordia. Distinctions 

 also exist between the sagitta, lapillus, and asteriscus, so com- 

 parative studies must be careful to properly identify the otoliths 

 examined. Variation in primordial form involves the size, shape, 

 and number per otolith. Surrounding the primordium (partic- 



