Asymmetrical individual both left and right otoliths of Tuna Scombrini collected from the Java Sea

Otolith can be used as a natural marker of fish stock or anomalies between individuals within fish stock. This research aims to study the relationships between fish length and otolith length and confirm differences in otolith shape between species in the Scombrini tribe and Carangidae family (Decapterus ruselli). Sample used in our analysis were collected around July - September 2023 from three different location, Rastrelliger brachysoma (Lekok waters, Pasuruan), Scomber australasicus (Bali Strait waters) and Decapterus ruselli (Sendang Biru, South Malang). Total 96 specimens were identified based on morphological character as Rastrelliger brachysoma, Scomber australasicus and Decapterus ruselli. Each of following samples were cut horizontally at the frontal head to open the skull and remove sagitta otolith below the hindbrain using dissecting set, then cleaned and stored dry in microtube 1,5 ml separately for left and right to further documented using Celestron Digital Microscope Pro Gray USB 44308. Otolith morphometric data collected includes maximum length using TpsDig and weight using analytical balance. The relationship between Forked Length (FL) and otolith length, length-weight relationship of otolith was determined using simple linear regression on software Statistical Product and Service Solutions (SPSS). Paired sample t-test was applied to determine asymmetrical size of left and right otoliths. In this study, it was found that the shape of the otoliths between species has differences in the rostrum, antirostrum, ventral and dorsal parts. FL-otolith length relationship of S. australasicus, R. brachysoma and D. ruselli are Y=0,725+0,164X, r2= 0,519; Y=0,191+0,230X, r2= 0,911; Y=3,156+0,075X, r2=0,071 respectively. The LW sagitta relationship shows that there is a very strong relationship (P<0,05) between L and W of otoliths. The results of the Paired Sample T-test showed that there was no significant difference in the size of the left and right otoliths (Sig>0.05). Otolith shape is influenced by fish behaviour, diet and environment and it may be used as additional information to distinguish between tribe or even species. Otherwise, its asymmetric relationship and LW relationship may be potential new methods for stock discrimination and have other implications such as stock assessment, understanding natural asymmetry, and its potential applications in fisheries management and conservation.


Introduction
Biological population analysis to observe annual growth variations or to classify the population from several specific variations, nowadays done with various research approaches, one of the approaches is using otolith.Specific character classify verifications for species level with otolith is still too finite, this condition caused otolith research somehow considered a lot of subjectivity [1].Otoliths of fish studies are expanded for ecological estimation, environmental stressors up to life-history processes.Several morphological parts such as otolith, scales, vertebrate up to statolith, can be used for completing and understanding many analyses as ages, shape and fish growth.Analysis of those morphological characteristics being important, also to understanding life-histories, population dynamics, demography and health of the population or species.Usage of Otolith for various species recently evolved rapidly, affected by analytical to technological advances and distribution as landmark discoveries [2].Morphological approaches to Teleost otolith studies, commonly used in biology, especially to assess several biological fish aspects such as auditory, movement, etc.Generally, abiotic factors such as food quality and quantity, temperature up to pH in the habitat, combined with biotic factors such as ontogeny, sex, size up to genetic, can influence otolith's morphology.More complex otolith morphological of a fish population or species, estimated to have higher intensity of mobility than fish population or species which has simpler otolith morphologic [3].
Scombridae family from Scombroidei suborder, is ones of important taxa from Pelagiaria clade.Taxas classification in general between and within Scombriformes order (Scombroidei was Perciformes before) is still finite and complex, including Scombridae family [4].Evolutionary histories of Scombridae family are still unclear, even though morphological and molecular approaches is used to solve the ambiguity.Complexity of Scombridae family and other Scombroidei members as Gempylidae and Trichiuridae, based on polyphyletical evidence within the taxon.Pelagiaria ambiguity strongly suspected occur caused of rapidly adaptive radiation between late Cretaceous and early Paleogene ages [5].Cryptic species within Scombridae family based on morphology as number of vertebrates combined with molecular analysis as phylogenetic, occur in Scomberomorus genera, especially for Scomberomorus guttatus complex [6].Potential cryptic species within Scombridae still high, and taxonomical solution to make this taxon clear still needed, based on morphologic, molecular up to ecological systematic analysis, including otolith morphology study.Otolith morphological analysis recently being ones of trusted effective method to classify inter and intraspecific variations [3].
Studies on two well-known Scombridae family as Java Sea fisheries products, blue mackerel (Scomber australasicus) and short mackerel (Rastrelliger brachysoma) are still lacking.Study with otolith analysis approaches of S. australasicus ever done on a population around New Zealand [7], and for R. brachysoma ever done on a population around Gulf of Thailand [8].Furthermore, otolith study on Scomber genera has been done on several species as Scomber colias from Atlantic [9], Scomber scombrus from North to Northwest Atlantic [10] and Scomber japonicus from North Pacific, around Japan State [11].Otolith study on Rastrelliger genera is also ever done on species as Rastrelliger kanagurta from Egyptian Red Sea and from Java to Bali Sea [12] [13].Limited study with otolith analysis approaches on S. australasicus and R. brachysoma from Java Sea, can be one of strong reasons to profiling this urgent field, remembering ecological degradation with weak fisheries regulation in Indonesia.Scombridae family members mostly are important fisheries commodities with high economical value and high demands from worldwide.Fisheries regulation management needs to be supported by systematic research data as from biological studies, the data can be the basis for management and conservation of commercial species as Scombridae [14].Profiling otolith morphology in this study, can be the basis for taxonomic and conservation approaches in the future, especially for S. australasicus and R. brachysoma in Java Sea.

Materials and Methods
Samples were collected around July -September 2023 from three different location, Rastrelliger brachysoma (Lekok waters, Pasuruan), Scomber australasicus (Bali Strait waters) and Decapterus ruselli (Sendang Biru, South Malang).All specimens were identified based on their morphological characters referring to the book by Carpenter & Niem vol. 3 -6.47 identified based on morphological character as Rastrelliger brachysoma, 32 identified as Scomber australasicus and 18 others identified as Decapterus ruselli.Forked Length (FL) from the most anterior point to the tip of the caudal fin was measured using a Fish Ruler, weight was measured using a digital scale with an accuracy of 0.1 g.In total 97 pairs of sagittae were removed through cutting the frontal head just above the eyes horizontally, then cutting the top of the skull until the scissors reach the spinal cord.The brain is removed to facilitate otolith removal which is just below the hindbrain.Otoliths are bones composed of calcium carbonate and a small number of organic matrices which are encased in a sacculus [15].All of otolith samples rinsed with detergent solution to remove tissue residue and bacteria, then stored dry separately for left and right in microtube 1.5 ml then documented used Celestron Digital Microscope Pro Gray 44308 and its maximum length as the dimensions from posterior to tip of the rostrum [16] were measured to the nearest mm used TpsDig.Individual otolith weight was determined using an analytical balance with readability 0.1 mg, for Decapterus ruselli, no weight data were collected due to lack of time.The normality test using the Kolmogorov-Smirnov asymptotic approach is applied to all combinations of data variables to validate the results of the linear regression test.The relationship between fish length and otolith length, length-weight relationship of otolith was determined using simple linear regression on software Statistical Product and Service Solutions (SPSS).Paired sample t-test was applied to determine asymmetrical size of left and right otoliths.

Results and Discussions
The forked length of Rastrelliger brachysoma ranged from 9,75 to 18,75 cm, Scomber australasicus ranged from 24,75 to 31,25 cm and Decapterus ruselli from 11,75 to 15,5 cm.The weight of Rastrelliger brachysoma ranged from 11 to 108 g, Scomber australasicus ranged from 225 to 407 g and Decapterus ruselli from 20 to 56 g.Otoliths are small calcified structures in the inner ear of a fish that grow incrementally over the lifetime of a fish, recording a timeline of information about the fish's biology and environment [17].Fish otoliths represent environmental information that can be used to reconstruct a history of the fish's movements, diet, habitat use, and environmental conditions in addition to its age.Several studies have shown a linear correlation between body length and otolith dimensions in various fish species can be described by a linear regression analysis.For Scombrini species (Rastrelliger brachysoma and Scomber australasicus), the results of the normality test on the variables FL (x) and Length of Left Otolith (Left_L) and Length of Right Otolith (Right_L) using an asymptotic approach stated that the data were normally distributed with asymptotic significance value more than 0.05 for each species.The results of linear regression state that there is a high correlation between forked length (FL) and otolith length (P > 0.05) in Scombrini species, presented in table 1.
The otolith length-weight relationship of Scombrini species is tested using linear regression and as shown in table 1, figure 1 and 2, both variables have a significant relationship.Graphs of FL-otolith length relationship of Scombrini species and Decapterus ruselli are given in figures 1 to 3. In contrast, the linear regression results of the relationship between FL and otolith length of Decapterus ruselli shown in table 2, state that there is no significant correlation between these two variables, either on the right or left otolith.The equation of FL-otolith length relationship is different for each side of otolith, but the results of two-way paired t-test as shown in table 3, the significance value for each species is more than 0.05.It can be concluded that the size of the right and left otoliths is the same for Scomber australasicus and Rastrelliger brachysoma, as is the case for the outgroup Decapterus ruselli.Another study by [12] on Sabre Squirrelfish (Sargocentron spiniferum) from the Southern Red Sea found that the relationship between otolith parameters and fish length can be affected by sex and season.otolith dimensions can be used as an index for environmental studies and for retrospective growth analyses in fish species [18] [19] regarding the relationship between fish length and otolith length, found that otolith size metrics were positively correlated with body length.This research also confirms the differences in otolith shape between species in Rastrelliger brachysoma, Scomber australasicus and Decapterus ruselli shown in figure 4 from a to c respectively.The guide of distinguished otolith morphology is included there.The differences in the shapes of the three are clearly visible in the rostrum and antirostrum.The Rastrelliger brachysoma otolith has one rostrum and one antirostrum and the posterior shape tends to be slightly rounded.In contrast, the Scomber australasicus otoliths tend to be square in shape and have one rostrum between two antirostrums.The otoliths of Decapterus ruselli have a shape that is very different from that of the Scombrini species, where the shape tends to be oval due to the convex dorsal and ventral parts, but also has an elongated antirostrum in the end of anterior.Study by [20] about otolith shape analysis is based on both shape index analysis and Fourier ellipse analysis, therefore, the description of the otolith shape and morphology of these three species can be used as a reference for future researchers to carry out otolith shape analysis as an additional classification of species, especially in the species Scomber australasicus and Scomber japonicus which is quite difficult to identify only from its morphological features, because the morphology of the two is quite identical.As for other ways to clarify species identification by [21] about otolith's of Scomberb using state that otolith shape analysis technique using the contour of otolith to classify the stock is considered more efficient than using morphological parameters.

Conclusion
There is a significant effect (P-value < 0,05) of the FL variable on otolith length in Scombrini species.
In the outgroup, Decapterus ruselli, it was found that there was no significant effect (P-value > 0,05) of FL on otolith length.Two-tailed paired t-test showed that there were no significant differences between left and right otolith's size for Scombrini species and Decapterus ruselli.Shape of otolith and size can be used as identifiers for species re-description.Otolith shape and its LW relationship may be the main variables.This information is required not only to provide baseline biological information for many important species, but also to enable the completion of a variety of studies, such as the analysis of the diet of species in the Java Sea, the identification of the fisheries resources utilised, and the reconstruction of ancient marine environments.

Figure 1 .
Figure 1.Graph of the FL-left otolith's length relationship (a), FL-right otolith's length relationship (b), LW relationship of left otolith (c) and LW relationship of right otolith (d) of Rastrelliger brachysoma.

Figure 2 .
Figure 2. Graph of the FL-left otolith's length relationship (a), FL-right otolith's length relationship (b), LW relationship of left otolith (c) and LW relationship of right otolith (d) of Scomber australasicus.

Table 1 .
Results of linear regression analysis of the relationship between forked length and otolith length of Scombrini (significance level was set at 5%).

Table 2 .
Results of regression analysis of the relationship between forked length and otolith length ofDecapterus ruselli (significance level was set at 5%).

Table 3 .
Results of paired sample t-test