Otolith Shape Analysis And Daily Growth Verification

What are Otoliths?

Otoliths are calcium carbonate growths located in the inner ears of bony fishes. Otoliths provide a fish’s balance, hearing ability, and velocity control. However, Ichthyologists can study otoliths to determine the age and health of fish just as dendrochronologists study tree rings.

Otolith background

Since Pannella described the existence of growth circles (otolith annuli) in fish otoliths in the early 1970s, research on the microstructure of otoliths has gradually increased. The daily growth of bony fish otoliths (sagittae) is considered a common phenomenon. The study of the microstructure of otoliths laid the foundation for hundreds of studies on early life history, age, growth, replenishment, migration, mortality, and population structure.

The growth model of the otolith microstructure is usually related to environmental conditions such as water temperature, salinity, and food supply. These environmental conditions will affect the broadening, contrast, and transparency of the otoliths. In addition, some early life history events can also be recorded on the otoliths, such as hatching, starting to eat, etc. The detection and analysis of the microstructure of otoliths can be used for the identification of fish populations, showing the feeding and growth history of fishes in the wild or underfeeding conditions for several days.

Article Context

Article topic: Otolith shape analysis and certification

Research object: European minnow (Squaliuscephalu)

Biological data: 200 larvae with body length between 6-91 mm and 0-180 days old otolith shape, growth data and body length data of larvae and juveniles

Analysis method: shape index; elliptic Fourier analysis; principal component analysis: canonical discriminant analysis; Kruskal-Wallis analysis of variance

Software used: SHAPE 1.3; R language

Scientific question: What is the relationship between the shape change of otoliths, the change of the sun and the growth?


The study of fish in the early life history stage is the basis for understanding their population dynamics. The relationship between age and growth can be used to assess the survival and recovery status of fish. The age of the fish can be judged by several calcified structures such as scales, operculum, vertebrae, fin rays, and otoliths.

Otoliths are usually the first calcification structure in the early development of fish. Some fish can already see three pairs (lapilli, sagittae, and asterisci) before hatching. Since Panella (1971) discovered the growth circles, otoliths have become a powerful tool for studying the early life history of fish.

Otoliths are calcium carbonate growths located in the inner ears of bony fishes and are related to hearing, balance, and acceleration control. Due to the different shapes and sizes of otoliths, the three pairs of otoliths are easier to distinguish. Otoliths are decellularized structures that are not dissolved or absorbed.

The microstructure of the otolith

The microstructure of the otoliths shows that the growth bands are pairs of concentric strips (translucent and opaque). The incremental growth of otoliths usually occurs at a constant rate through the different deposition of calcium carbonate and protein (Cross section view of otolith).

The morphology of otoliths varies greatly among species, but they are also affected by environmental conditions. Therefore, they show both inter-species and intra-species differences.

The shape and structural characteristics of otoliths should be concerned because their growth is related to habitat characteristics (such as water temperature, water flow, salinity, food supply) or environmental pressure, and is related to major life-history events in the first year, such as hatching, first time eating, the metamorphosis from larvae to juveniles, or the transition to a specific life history are also connected to a certain degree. There are certain changes in the shape of fish otoliths throughout the development process, but the degree of its relationship with the development process is rarely studied.

Validation of the otolith deposition rate is an important prerequisite for accurately estimating the age of larvae and juveniles using otoliths. This verification can be achieved by growing larvae and juveniles of known age under controlled laboratory conditions, using fluorescent markers for statistical inference, or analyzing marginal increments. Among them, the most reliable method to obtaining the daily cycle of otoliths is to analyze the otoliths of larvae and juveniles by artificial growth in a similar natural environment.

European Minnow (Squalius cephalus)

Squalius cephalus is one of the most common species in freshwater in Europe. Because of its high environmental tolerance (such as a large thermal tolerance range) is also very abundant in environments where human factors are severely disturbed. However, there are no reports about the early sun rings (very thin ring of line) and annuli of otolith of this species. Therefore, the number of otolith daily increments that appeared during the hatching and the formation time and rate of the first increments are still unknown.

In this study, the larvae and juveniles of this species artificially cultured in the laboratory were used to verify the otolith deposition rate. The morphological changes of otoliths during the ontogeny were studied to analyze the relationship between age and shape (and to some extent growth), as well as the formation time of the first sun ring and the deposition rate of otoliths.

Research result

1. Otolith development

Only micro otoliths (Lapillus) and sagittal otoliths (Sagitta) appear during hatching, and stellate otoliths (Asteriscus) appear after hatching 20-30. The morphology of the three pairs of otoliths changed during the ontogeny.

Micro otoliths and sagittal otoliths were round when hatched and remained round for 20 days. During the development period, both pairs of otoliths showed obvious primordia and sun rings, which were difficult to distinguish. These are distinguished by their position on the head.

The front part was the micro otolith, and the rear part was the sagittal otolith. Early otoliths are slightly larger than micro otoliths. After 30 days of hatching, stellate otoliths appeared, and all three pairs of otoliths were oval. At 45 days after hatching, the size and shape of the micro-otoliths did not change much, while the sagittal otoliths and stellate otoliths became significantly longer, rounded, and larger. 45 days after hatching, the rear side of the micro-otoliths has a square edge, while the front side is still round.

The sagittal otolith is round in the front and pointed at the back. Micro-otoliths and sagittal otoliths increase at a faster rate in the front-to-back direction, while the dorsal-ventral axis of stellate otolith grows faster and is rounder. After 60-180 days of hatching, the rear part of the micro-otolith is still square, the sagittal otolith is elongated on the front and back axis, and the back is also thin and fragile, and the edge of the stellate-otolith becomes an irregular shape, similar to serrations.

Since stellate otoliths appear 20 days after hatching, they are not suitable for measurement and fixation. The sagittal otolith showed an obvious micro-increase in the early stage, but it became too fragile to be operated in the later stage. When it was hatched 45, it often broke during the extraction process and was not suitable for further analysis. Therefore, only consider the further analysis of micro otoliths.

2. Micro-otolith morphology analysis

Micro-otolith analysis results show that the shape of otoliths will change with age. Compared with the Fourier transform method, the difference between the otolith age groups verified by the shape index method is not obvious.

The CDA (canonical discriminant analysis) data of the shape index divides the age into three groups 0-30 dph (dph: days post-hatching), 45-90 dph, and 120-120 dph), and the CDA data of the Fourier coefficient divides the age into 5 Group (0-30 dph, 45 dph, 60 dph, 90 dph, and 120 to 180 dph).

Since the Fourier transformation gives more obvious discrimination, more attention will be paid to this method in the following research. Therefore, the age group is divided into five stages: the youngest (<30 dph), the oldest (>120 dph) and three middle age groups (30-45 dph, 45-60 dph and 60-90 dph).

In order to ensure that the changes in the shape of the otoliths observed are related to the age and not to the growth (size) of the fish, this research also performed another analysis of the difference between the fast-growing and slow-growing otoliths.

In each age group, 4 groups were created: two groups of fast growth and slow growth were analyzed based on the body length of the fish, and two groups of fast growth and slow growth were analyzed based on the daily growth rate of otoliths. The results showed that there was no significant difference in otolith morphology between the slow-growing group and the fast-growing group.

3. Micro-otolith deposition rate

A black spherical primordium can be seen in the center of the micro otoliths when they are just hatched. Although the otoliths are formed, there are no sun rings. At 10 days after hatching, the otoliths were dark and 10-day bands appeared, indicating that the first slight increase corresponds to the hatching, and day increments were formed every day. The primordium radius (from primordium to hatching mark) of micro otoliths is 18.05-35.47um (mine ± SD = 24.48 ± 3.6um).

The calculation range of the micro-otolithic sun rings is 0-120 days, as shown in the linear regression equation (y=101x-0.469, R²=0.999), the intercept and 0 (t-test, t =−0.686, P> 0.05) and the slope It is not significantly different from 1 (t-test, t = −0.005, P> 0.05).

4. Otolith growth

The increment width around the primordium (otolith nucleus) was less than 2.4µm on average and dropped to less than 2µm, but after a breakpoint occurred between 17 and 20 days, the increment width increased to more than 2.8µm, and then slowly dropped to the otolith edge.


This experiment is based on the artificially cultured European minnow micro-otolith shape and microstructure research. It shows that the shape of the otolith changes significantly with individual development. Further research found that the first deposition of otoliths corresponds to the hatching, and the first-day increment corresponds to the day of hatching, after which one daily increment appear every day.

The change of otolith shape will be affected by both exogenous factors and endogenous factors, and there is a growth and development process. Micro-otoliths appear after 20-30 days of hatching, and the edges of sagittal otoliths become fragile in the later stages of development and are not suitable for longer-term microstructure studies. This is a common phenomenon in carps.

This research has supplemented the age and otolith growth information of European minnow, which can increase the understanding of its biology. It provides necessary data for future research on the determination of the age of European minnow under similar environmental conditions, resource supplementation, and the impact of its growth on the environment.