The developmental variations of the sagitta otolith in the young and mature male of a hermaphrodite polynemidae fish, Eleutheronema tetradactylum (Shaw, 1804)

The Eleutheronema tetradactylum is a protandrous, hermaphrodite, marine perciformes fish. The body length of this fish acts as an important diagnostic marker for male and female discrimination. The present study describes for the first time the ultrastructural characteristics on the medial surface of the sagitta otolith in different body size groups of males of E. tetradactylum (Polynemidae: Perciformes) using scanning electron microscopy. The sagitta is a spindle-shaped structure that includes a well-developed rostrum and a poorly developed antirostrum. The sulcus is ostio-pseudocaudal type, almost straight and devoid of the collum. The ostium is a well-developed, vase-shaped structure. The cauda includes the colliculum and a well-developed caudal bulb with several distinct growth stripes. The length of the caudal bulb is significantly correlated to the growth of the body size of the fish. The excisura major is indistinct and the excisura minor is absent. The cristae are distinct on both sides of the sulcus. The one-way ANOVA test revealed that the development of several sagitta features shows significant differences in various body size groups of E. tetradactylum. The growth of the sagitta length is more closely related to the fork length than the sagitta width. Therefore, the sagitta length and the caudal bulb length can be used as important predictors to evaluate the fish size. The cauda region of the sagitta in E. tetradactylum is unique as well as more decorative than those of another Polynemidae fish and other hermaphrodite, marine perciformes fishes. The sagitta characteristics of E. tetradactylum might be advantageous in the identification of the sex and the taxonomy of the hermaphrodite fish species.


INTRODUCTION
The otolith is a calcareous anatomical structure in the inner ear of fish and assists in the sensation as well as the body balancing by stimulation of inner ear hair cells (Sanchez & Martinez, 2017). In the taxonomic field, the fish species identification is significantly supported by the structural characteristics of the otolith due to its longer time of degradation (Rodríguez Mendoza, 2006;Vilizzi, 2018;Mitsui et al., 2020). The otolith includes one pair of sagittae, asteriscii and lapilli (Popper et al., 2005). The sagittae are the largest pair in size in fish groups (except, order Siluriformes and Cypriniformes) and are included in several species-specific characteristics (Harvey et al., 2000;Kontaş & Bostanci, 2015;Yilmaz et al., 2015;Mehanna et al., 2016). The sagitta has two surfaces which are the lateral (outer) and medial surface (inner). The medial surface of the sagitta is well-decorated with different morpho-structural features (Smale et al., 1995;Jawad et al., 2018;Bardhan et al., 2021), which characteristically varies with the fish groups and various environmental factors in their respective habitats (Kumar et al., 2012;Omar & AMohamed, 2016;Abdulsamad, 2017;Sanchez & Martinez, 2017;Jawad et al., 2018;Pracheil et al., 2019). Several studies have dealt with the morphological descriptions of the sagitta of marine Perciformes fishes (Hunt, 1992;Smale et al., 1995;Gierl et al., 2018;Jawad et al., 2018), but no ultrastructural studies on the sagitta are available in the hermaphrodite perciformes fishes. Additionally, the perciform family Polynemidae have eight genera but the sagitta morphologies are only described by light micros-copy in only Polydactylus virginicus (Santificetur et al., 2017).
The E. tetradactylum (Polynemidae: Perciformes) is in fact a protandrous, hermaphrodite marine fish (Patnaik, 1967;Kowtal, 1972;Stanger, 1974;Motomura, 2004;Sadovy & Liu, 2008). They act as functional male in their early part of life and then act as functional female for the later periods of life (Patnaik, 1967;Kowtal, 1972;Stanger, 1974;McPherson, 1997;Ballagh et al., 2012). The body size (fork length) of E. tetradactylum act as an important diagnostic marker for the male and female discrimination among the individuals (Kailola et al., 1993;McPherson, 1997;Ballagh et al., 2012). It is reported that the fork length of the fish around 24 cm act as a mature male and greater the body length acts as female (Stanger, 1974;Kailola et al., 1993;Motomura, 2004). Ballagh et al. (2012) briefly described the sagittae of Eleutheronema tetradactylum in a study of the relationship between the age and the growth of total body length of this fish. It has been stated that the development of the sagitta components (i.e., sulcus, ostium, rostrum, etc.) is associated with the body size (length/weight) groups of the sexually dimorphic fishes (Harvey et al., 2000;Jawad et al., 2018;Bardhan et al., 2021). However, there is no information regarding the developmental variations of the otolith morphologies among the young male and the mature male of the protandrous, hermaphrodite fishes. The present study aims to investigate detail ultrastructural characteristics of the sagitta otolith of the protandrous Eleutheronema tetradactylum (Shaw, 1804). A comparative developmental relationship of the various sagitta constituents between the young male and the mature male groups is conducted here. The results of the E. tetradactylum are also compared with the available data on the sagitta for a protandrous Polynemidae fish (Polydactylus virginicus) and three protandrous Sparidae fishes (Sparidentex hasta, Acanthopagrus berda, and Acanthopagrus latus).

Sample collection and grouping
A total of 130 individuals of Eleutheronema tetradactylum (Shaw, 1804) (Polynemidae: Perciformes) were randomly collected from the fish market of Kolkata, West Bengal, India. The samples were identified by the Zoological Survey of India (ZSI), Kolkata, West Bengal, India. The body size of the individuals was examined by their fork length (i.e., the length from the anterior tip of the longest jaw to the median point of the caudal fin) (Önsoy et al., 2011;Butler et al., 2021) and was measured using a centimetre scale. The specimens were divided into four groups according to their fork length of the fishes (FL) (Jawad et al., 2011): group I (Gr-I), 11-12 cm (Mean: 11.55 ± 0.30), n = 25; group II (Gr-II), 15-16 cm (Mean: 15.54 ± 0.30), n = 45; group III (Gr-III), 19-20 cm (Mean: 19.58 ± 0.31), n = 35; group IV (Gr-IV), 23-24 cm (Mean: 23.52 ± 0.31), n = 20. The specimens in group IV were mature males and the individuals in other three groups were younger males.

Collection of the sagitta otolith and scanning electron microscopy
One pair of sagittae were removed from the saccule of the inner ear of each individual of the four groups (Ruck, 1976;Jawad et al., 2018), cleaned with water and 70% ethanol, and stored dry in individual plastic tubes.
For ultrastructural studies on the medial (inner) surface of the sagittae, the right sagitta was examined . The sagittae were dried and mounted on an aluminium stub using double-sided carbon tape. The sagittae were gold-coated by DWARDS, RV5 coater, and analysed in an EVO18, ZEISS.
In the work, all the terminologies used for the description of the structural constituents of the medial surface of the sagitta are following Smale et al. (1995), Jawad (2007) and Bardhan et al. (2021).

Morphometry and statistical analysis
The measurements (mean value ± SD) of the various sagitta constituents were taken for the sagittae from the four size groups using image-processing software "ImageJ 1.51t" (Wayne Rasband, NIH, USA). The weights of sagittae were taken with a digital weight machine (Mettler Toledo ME204). A normality test using Shapiro-Wilk test was applied to check the distributions of the studied sagitta constituents among the four groups. The test met the assumption of parametric analysis and a one-way ANOVA followed by Tukey's test was performed using XLSTAT statistical program to determine the statistical significance (P < 0.05) on the developmental differences on the growth of the studied sagitta features in the different life stages (young males to mature male) of E. tetradactylum.

General morphology of the sagitta
The sagitta of E. tetradactylum males is an oblong or spindle-shaped structure (Figs. 1, 2A-D). The medial surface of this sagitta is slightly convex and enriched with different structural features (Figs. 1, 2A-D, Tables 1-2). The ultrastructural characteristics of the various constituents on the medial surface of this sagitta is described with the following points.
The sulcus lacks the collum (Figs. 1, 2A-D). A distinct 'V'-shaped ridge is developed near the junction of ostium and cauda in the sulcus groove (Figs. 1,.

The rostrum and antirostrum
The rostrum part of the sagitta is well-developed while the antirostrum part is comparatively very shorter than that of the rostrum (Figs. 1, 2A-D, Tables 1-2). A distinct gap is developed between the rostrum and the antirostrum, termed as excisura major (Figs. 1, 2A-D, Tables 1-2).

The margins and surface sculptures
The sagitta is dorsally oval-shaped with a distinct irregular margin and is ventrally slightly curved with sinuate margin (
in body size groups (Figs. 2A-D; Tables 1-2). The sagitta length and width vary accordingly with the four body size groups, while the sagitta weight remains the same after certain growth of body size (Fig. 3). In the study, based on the body size, the fishes in group IV (Gr-IV) are comparatively older (mature male) and those in group I (Gr-I) is relatively younger (younger male) among the four studied groups. All the studied sagitta components except the otolith width, rostrum length, antirostrum length, sulcus length and the width of crista superior-dorsal margin show a significant difference between these two groups (Table 2). Furthermore, some of the sagitta constituents such as the caudal length (Cl, Fig. 4A), length of caudal bulb (Cbl, Fig. 4B), width of crista inferior-ventral margin (CriV, Fig. 4C), sulcus width (SW, Fig. 4D), and sulcus depth (SD, Fig. 4E) show a relative developmental variation in the four different body size groups ( Table 2). The normality test shows that all the studied sagitta features are normally distributed in the four body size groups (Table 3) Table 2). The growth stripes in different parts of the sulcus are prominent in the groups with larger specimens (Table 1). It is observed that the margins and marginal sculpture of the sagittae are var-  ied with the increment of the total body length of fishes (Figs. 2A-D; Table 1). The outer margins (i.e., dorsal and ventral margins) and their marginal sculptures (i.e., smooth margin, irregular margin, etc.) of the sagittae are varied with the increment of the total body length of the fishes (Figs. A-D; Table 1). The dorsal margin of the sagitta in the individuals in group I is mostly smooth while it is developed as characteristically irregular in the groups of the larger specimen (Figs. 2A-D; Table 1). The ventral margin of the sagitta in the groups (i.e., Gr-II, Gr-III, and Gr-IV) with larger specimens are the sinuate type with very distinct several marginal indentations which are very indistinct and few in the sagitta of the individuals in group I (Figs. 2A-D; Table 1). The groove on the mid-dorsal mar-gin of sagittae is absent in group (Gr-I) with smaller specimens while it is characteristically very distinct with various shapes and sizes in groups (Gr-II, Gr-III, and Gr-IV) with larger specimens (Tables 1-2). A 'V'-shaped ridge is developed near the junction of ostium and cauda in the sulcus and is well-developed in groups with larger specimens (Asterisk, Figs. 2A-D; Table 1). Development of the caudal bulb (Cbl) at the caudal end is significantly and positively correlated to the increment of the total body length ( Figs Table 2). The development of the sagitta     length is significantly increased with the growth of total body length of fishes (Figs. 3, 5A; Table 2). The sagitta width is slightly increased with the growth of total body length and their developmental relationship is not significant ( Fig. 5B; Table 2). The sagitta weight is initially increased with the increment of the total body length and after a certain body size (19-20 cm FL), its development remains the same or insignificantly developed as body length increases (Figs. 2A-D, 3, 5C; Tables 1-2). The development of the caudal length (Cl) and sulcus width (SW) is negatively correlated to the total body length (Figs. 4, 5E, 6F; Table 2). The sulcus depth (SD) and the width of the crista inferior to the ventral margin (CriV) are larger in groups with smaller individuals than those in groups with larger individuals (Figs. 2A-D, 4, 5E, 8A; Table 2). In the present study, it is observed that the sagitta morphologies of male E. tetradactylum have several relative relationships in respect of hermaphroditism with another protandrous Polynemidae fish (Polydactylus virginicus) and three protandrous Sparidae fishes (Sparidentex hasta, Acanthopagrus berda, and Acanthopagrus latus) irrespective of their male/female discriminations (Table 4). This comparative study showed that the sagitta morphologies are closely similar, but with some significant species-specific differences within the Polynemidae fishes while they are characteristically different among the species of other protandrous Perciformes fishes ( Table 4). The morphostructural and morphometric analysis advocates that the growth of the sagitta length and caudal bulb length are significantly increased with the increment of the total body length than that of the sagitta width and weight (Figs. 3, 5A-C, 7B; Table 2). The sagitta length, weight, and caudal bulb length may be used as important predictors to evaluate the body size of E. tetradactylum.

DISCUSSION
The detailed morphostructural characteristics of the medial surface of the sagittae of the male E. tetradacty- lum (Polynemidae: Perciformes) in different body size groups are described for the first time using scanning electron microscopy. The development of the sagitta constituents varies in different stages of the sexual maturity of the male E. tetradactylum. This kind of developmental differences in different body size groups are also reported in other Perciformes fishes; i.e., Chlorurus sordidus (Jawad et al., 2018), Anabas testudineus , Umbrina cirrose (Başusta & Khan, 2021). The characteristics of various sagitta constituents of E. tetradactylum slightly differ from those of the other Polynemidae fish (e.g., Polydactylus virginicus, Santificetur et al., 2017) and considerably differ from other protandrous marine Peciformes fishes (e.g., Sparidentex hasta, Acanthopagrus berda, and Acanthopagrus latus; Abdulsamad, 2017) ( Table 4). The sagitta of E. tetradactylum is a spindle-shaped structure, however this varies with the protandrous Polydactylus virginicus and the three protandrous Sparidae fishes (Table 4). The medial surface of the sagitta comprises a well-developed sulcus groove as also reported in other fishes (Dehghani et al., 2016;Omar & Moselhy, 2016;Abdulsamad, 2017;Jawad et al., 2018;Khedher & Fatnassi, 2018), and this groove may assist for connecting the medial surface with the sensory cells of the internal ears (Popper & Hoxter, 1981;Popper & Lu, 2000). A number of sulcus morphologies of E. tetradactylum are characteristically identical with another marine as well as freshwater Perciformes fishes (Smale et al., 1995;Bremm & Schulz, 2014;Omar & AMohamed, 2016;Omar & Moselhy, 2016;Abdulsamad, 2017;Jawad et al., 2018;Bardhan et al., 2021). Hunt (1992) stated that the otolith morphologies between male and female fishes are almost the same, whereas several authors reported that there are some structural differences between these genders in many fish species (Vallisneri et al., 2008;Bostanci et al., 2012;Kontaş & Bostanci, 2015). In the present study it is observed that the development of sagittal constituents varies also with different body size groups in a particular gender (e.g., male fish).
The development of the medial surface sagitta structures on of E. tetradactylum is characteristically variable among different body-size groups, as described in other fishes (Jawad et al., 2018;Bardhan et al., 2021). However, several sagitta features of E. tetradactylum and Polydactylus virginicus (Santificetur et al., 2017) are mostly similar (Table 4) and possibly the common identifying features of the sagitta in the Polynemidae species. In the present study, we show that the sagittae of the smaller fish group possess entire smooth surface and marginal sculpture, whereas those sagitta features considerably differ with the increase of the total body length, probably due to various pattern of the calcium carbonate crystals deposition (Campana & Thorrold, 2001;Schwarzhans & Grenfell, 2002;Volpedo & Echevarria, 2003;Vilizzi, 2018;Pracheil et al., 2019).
It has been suggested that the increase of the otolith weight is significantly proportional to the total body length of the individuals in some fish groups but the length and width of the otolith are not (Gümüs & Kurt, 2009;Bardhan et al., 2021). In the present study, the weight of the sagitta in the E. tetradactylum is increased with the growth of total body length of the individuals in the young male groups with smaller body size (i.e., the sagitta weight in group I fishes is lesser than that in the group II), while this increment of the otolith weight is restricted in the mature male group with larger body size (i.e., the sagitta weight in the group III fishes is almost similar to that in the group IV). Furthermore, the morphometric measurements of the sagitta in E. tetradactylum reveal that the length of the otolith is significantly related to the total body length of the fishes instead of the otolith width and also reported in other fish families such as Nototheniidae (Lombarte et al., 1991), Merluccidae (Lombarte & Lleonart, 1993); Labridae (Skeljo & Ferri, 2012); Cyprinidae (Kontaş & Bostanci, 2015), Sparidae (Khedher & Fatnassi, 2018), Sciaenidae (Carvalho et al., 2020). It is assumed that the sagitta formation in fishes may be completed at a certain body length due to constant weight. Additionally, the development of the caudal bulb is directly proportional to the growth of total body length in the E. tetradactylum. It is presumed that the relationship between the total body length and various characteristics of the otolith may be varied with the fish species and their relative habitats (Jawad, 2007;Jawad et al., 2018;Khedher & Fatnassi, 2018;Bardhan et al., 2021).
The sagitta features of the male E. tetradactylum shows some characteristic similarities with other marine perciformes fishes irrespective of their male/female gender specificity (Ballagh et al., 2012;Kontaş & Bostanci, 2015;Avigliano et al., 2016;Omar & AMohamed, 2016;Omar & Moselhy, 2016;Santificetur et al., 2017;Abdulsamad, 2017;Khedher & Fatnassi, 2018;Jawad et al., 2018). In the present study, sagitta characteristics of the male E. tetradactylum are described and may have some variations with those of the female individual but this requires further investigations. The results of the current study advocate that the sagitta features in different maturation phase of maleness of E. tetradactylum may be convenient for future studies of the otolith of other protandrous, hermaphrodite fishes and ultimately find out the relatedness among the species as well as male and female discriminations of the Polynemidae family in respect of systematics and gender choice respectively.

ACKNOWLEDGMENTS
The authors thank principal Prof. Mohua Das, subject coordinator Prof. Debarati Mukherjee, and all other faculty members of the Department of Zoology, Women's College Calcutta, Kolkata, West Bengal, India, for their help in this study. The authors are grateful to all members of SEM Unit, Zoological Survey of India (ZSI), Kolkata, for their help with the scanning electron microscopy studies. The authors are thankful to Dr. Amit Mukhopadhyay, Mollusca Section, Zoological Survey of India (ZSI), Kolkata, for his constructive suggestions in this work. The authors are also thankful to the Fish Division, Zoological Survey of India (ZSI), Kolkata, for the identification of the studied fish in this work. The authors are also grateful to the reviewers and editorial team of the manuscript for their constructive suggestions.

AUTHORS' CONTRIBUTIONS
S.R.: Design the work, dissection of first part of the otolith, guidance of dissection, SEM preparation and photography, arrangement and analysis of figures, illus- Roy, S. & Bardhan, I.: Ultrastructure of the sagitta of the male E. tetradactylum (Shaw, 1804), in different body size groups Pap. Avulsos Zool., 2021; v.61: e20216178 10/12 tration steps and microscopy, statistical analysis, graphical representations, and part of the description, supervision on the whole work, final revision of the whole manuscript. I.B.: Collection of the samples, dissection of the sagitta otolith from fish, schematic drawing of the otolith, SEM preparation, morphological and morphometry data collection, arrangement of text, table and references.