Investigating otolith mass asymmetry in six benthic and pelagic fish species (Actinopterygii) from the Gulf of Tunis

Otolith mass asymmetry can significantly affect the vestibular system functionalities; usually, the X values of mass asymmetry vary between −0.2 and + 0.2 (−0.2 < X < + 0.2). These values can change during a fish life and therefore they are not related to the fish total length. We collected a total of 404 fish specimens from the Gulf of Tunis, including three pelagic species: Sardina pilchardus (Walbaum, 1792) (74 otolith pairs), Trachurus mediterraneus (Steindachner, 1868) (66 otolith pairs), and Chelon auratus (Risso, 1810) (60 otolith pairs) and three benthic species: Gobius niger Linnaeus, 1758 (77 otolith pairs), Mullus barbatus Linnaeus, 1758 (60 otolith pairs), and Trachinus draco Linnaeus, 1758 (67 otolith pairs). The relation between the total length and the otolith mass asymmetry was first calculated and compared, and then was evaluated. The comparison of the otolith mass asymmetry between benthic and pelagic species showed a significant difference (P < 0.05), where the absolute mean value of X does not exceed the critical value (0.2) for all the studied species. No relation has been found between the magnitude of the otolith mass asymmetry and the length in both benthic and pelagic specimens. Environmental factors have an indirect effect on somatic growth and otolith accretion. The significant difference found in this study can be due to the difference between the benthic and pelagic environments.


Introduction
Otoliths are calcified structures found in the inner ears of teleosts, in the vestibular system. Every fish has three otolith pairs mostly composed of calcium and carbonate layers precipitated in an organic matrix (Carlström 1963;Panfili et al. 2002;Pracheil et al. 2019). These paired structures are involved in acoustic and balance systems of teleosts (Paxton 2000). These calcified structures are metabolically inert, they grow by accretion and they cannot be used by the organism as a source of calcium (Campana and Neilson 1985). Thanks to these properties, otolith pairs and especially the sagittas are used to rebuild fish life cycle and their interactions with the environment (Vignon and Morat 2010).
For many years, otolith shape has been routinely used to differentiate between fish stocks (Keating et al. 2014;Bailey et al. 2015;Ider et al. 2017;Mejri et al. 2018b;Nazir and Khan 2019;Avigliano et al. 2020). Otolith microchemistry has also been used to explore the environmental and food effects on fish metabolism (Mille et al. 2015;Perry et al. 2015). Furthermore, many presently cited studies indicate that the otolith mass asymmetry is also an important parameter because otoliths play a major role in acoustic functionalities.
The inconsistency between the right and left otolith's movements inside the inner ear can cause balance problems and sound perception difficulties (Lychakov and Rebane 2005;Lychakov et al. 2006).
Several studies on otolith mass asymmetry have shown that the majority of the symmetric fish species have X values within the range of -0.2 < X < +0.2 Rebane 2004, 2005;Jawad 2013). Thus, in theory, only an absolute value of X that exceeds 0.2 can alter the acoustic functionality of a fish (Lychakov et al. 2006). Moreover, the relation between otolith mass asymmetry and fish total length has been explored (Mejri et al. 2018a). Lychakov and Rebane (2004) have shown a relation between saccular otolith mass asymmetry and fish length exists only in littoral and bottom populations and not in the pelagic species. Also, some studies have shown that there is no relation between otolith mass asymmetry and fish total length (Jawad 2013;Yedier et al. 2018). The presently reported study aimed to compare the otolith mass asymmetry between pelagic and benthic species and to assess the relation between mass asymmetry and the fish total length. The following species were studied: three pelagic species: Sardina pilchardus (Walbaum, 1792), Trachurus mediterraneus (Steindachner, 1868), and Chelon auratus (Risso, 1810) and three benthic species: Gobius niger Linnaeus, 1758, Mullus barbatus Linnaeus, 1758, and Trachinus draco Linnaeus, 1758.

Sample collection
A total of 204 benthic fish species and 200 pelagic fish samples were collected from the Gulf of Tunis, in the north of Tunisia (36°49′09′′N, 10°18′22′′E) from March to May 2017 ( Fig. 1). In this study, we only used adult fishes to compare the otolith mass asymmetry between benthic and pelagic species without considering gender. Standard and total length (L s , L t ) were measured for each specimen to the nearest mm. The mean total length of the benthic species ranged from 150.8 ± 14.34 for Mullus barbatus to 238.322 ± 16.59 for Gobius niger and from 150.375 ± 9.69 to 235.14 ± 11.62 for pelagic species (Sardina pilchardus and Chelon auratus, respectively) ( Table 1).

Otolith extraction
Sagittal otoliths pairs were manually removed by the dissection of the auditory capsules, washed with distilled water, and air-dried at room temperature. The weight of the right and left paired otoliths was also determined using a precision electronic balance (Mettler Toledo AL204) to an accuracy of 0.1 mg (Table 1).

Data calculation
The otolith mass asymmetry (X) was computed using the following formula: where M R and M L are the otolith masses of the right and left paired otoliths, and M M is the mean mass of the right and left paired otoliths.  Theoretically, X values vary from −2 to +2. These limit values indicate maximal asymmetry while the '0' value refers to the absence of asymmetry between right and left otoliths of the same fish. A negative value of X means that the left otolith is heavier than the right one (M L > M R ), whereas a positive value of X means the opposite.
The relation between absolute otolith mass asymmetry (│X│) and the total fish length was calculated using the following formula: where a is the coefficient characterizing the growth rate of the otolith and b is a constant for a given species.

Statistical analyses
The comparison of the otolith mass asymmetry between benthic and pelagic specimens was performed using Student's t-test. To assess the existence of a relation between the total length and the absolute otolith mass asymmetry, a regression analysis was used to calculate correlation coefficients and regression equations for each species. XL-STAT (2007) software was used for all statistical analyses.

Results
It is known that the absolute symmetry between left and right otolith (X = 0) is rare, usually, X values fluctuate around 0. In this study, the percentage of asymmetry exceeded 50% in all the studied fish species. Trachinus draco, which is a benthic species, has shown an asymmetry rate equal to 100%. This means that all the otolith pairs of the 67 used fish samples are asymmetric in terms of weight, unlike the results observed in Sardina pilchardus population which have shown the lowest asymmetry rate with only 53%.
The otolith mass asymmetry was within the range of −0.496 ≤ X ≤ 0.3379 for all the studied species. It varied between −0.0513 and 0.1531 for benthic species and between −0.496 and 0.3379 for pelagic species (Table 2). The mean values of │X│ were calculated for all inves-tigated species. The results have shown a mean value of │X│ equal to 0.0251 ± 0.0021 for benthic species and 0.0383 ± 0.0046 for pelagic species. Moreover, a significant difference was found between benthic and pelagic fish species (P < 0.05) ( Table 3).
The correlation coefficients R 2 and regression equations were calculated for all of the six studied species each apart. The results reject the hypothesis since no significant relation between absolute otolith mass asymmetry and the total fish length was found (0.0008 ≤ R 2 ≤ 0.0356) (Table 4).

Discussion
In the presently reported study, all benthic and pelagic species had a mean value of otolith mass asymmetry varying from −0.2 to 0.2 similar to previous studies (Lychakov et al. 2008;Jawad 2013;Jawad and Sadighzadeh 2013). Only pelagic species showed X values exceeding the critical limits, in particular, T. mediterraneus with an otolith mass asymmetry −0.3665 < X < 0.3379. This is probably related to the physiological state of this species, its habitat, and environmental factors (abiotic and biotic) as previously reported by Grønkjaer (2016) and (Izzo et al. 2018) since the fish studied survived at different latitudes and longitudes. In the same context, the variations of environmental factors as well as anthropogenic ones have remarkable effects on the development of otoliths (Munday et al. 2011). In fact, in their study on the physicochemical parameters of the Gulf of Tunis, Ben Lamine et al. (2011) showed that this area suffers from several problems, such as metal pollution and urban discharges. Likewise, other studies have shown   that pollution can affect the growth of otoliths (Elsdon and Gillanders 2002;Munday et al. 2011;Perry et al. 2015). It has been shown that metals impact otolith growth when fish inhabiting affected areas are likely to accumulate metals, including anthropogenic metals from surrounding environments, and transfer / transport them to the upper links in the food chain (Wang 2002). This is reflected in the continued incorporation of the metals into the crystal matrix of the otolith since they are metabolically inert and their increments can undergo resorption. Our results are consistent with other studies conducted on the effect of metal accumulation on otolith growth (Vrdoljak et al. 2020).
Morphological variability of sagittae is impacted by a dual regulation: genetic and environmental factors (L'Abée-Lund 1988; Lombarte et al. 2010;Vignon and Morat 2010;Annabi et al. 2013). Also, the increase or decrease in otolith mass asymmetry can negatively affect other factors that are necessary for the life of the fish, especially the sense of hearing and balance. The otolith mass asymmetry has been used as a bioindicator to test the condition in different aquatic habitats (Grønkjaer 2016) and to test different environmental effects on fish populations. In our study, we have shown that the relative size of sagittae is larger in benthic species than in pelagic ones, which could be related to the different ecological niches of the studied fish (Lombarte and Cruz 2007).
The relation between the otolith mass asymmetry and the total length has been investigated in several studies (Mille et al. 2015;Yedier et al. 2018). We used the absolute value of X to prove whether there is any relation between the absolute otolith mass asymmetry and fish total length. According to the presently reported results, the absolute value of the otolith mass asymmetry does not depend on the total length in any studied species. Our results agree with the previous findings which investigated roundfish and flatfish (Jawad 2013;Yedier et al. 2018).
The otolith mass and shape asymmetry was explored in many studies all over the world. In Tunisia, otolith research is limited to fish stock identification and the assessment of sexual dimorphism. We need more research studying the impact of the environment on otolith mass asymmetry and on fish behavior. Therefore, it is important to use a large number of specimens and a wide range of body sizes in future studies to fully understand the relation between the asymmetry in otolith mass and the fish length.

Conclusion
Environmental factors have an indirect effect on somatic growth and otolith accretion. The significant difference found in this study can be related to the difference between the benthic and pelagic environments. Food and genetic variability can also be used to explain the presently reported results.